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The thesis entitled „Investigations on the significance of nucleo-cytoplasmic transport for the biological function of cellular proteins" aimed to unreveal molecular mechanisms in order to improve our understanding of the impact of nucleo-cytoplasmic transport on cellular functions. Within the scope of this work, it could be shown that regulated nucleo-cytoplasmic transport of a subfamily of homeobox transcription factors controlled their intra- and intercellular transport, and thereby influencing also their transcriptional activity. This study describes a novel regulatory mechanism, which could in general play an important role for the ordered differentiation of complex organisms. Besides cis-active transport Signals, also post-translational modifications can influence the localization and biological activity of proteins in trans. In addition to the known impact of phosphorylation on the transport and activity of STAT1, experimental evidence was provided demonstrating that acetylation affected the interaction of STAT1 with NF-kB p65, and subsequently modulated the expression of apoptosis-inducing NF-kB target genes. The impact of nucleo-cytoplasmic transport on the regulation of apoptosis was underlined by showing that the evolutionary conservation of a NES within the anti-apoptotic protein survivin plays an essential role for its dual function in the inhibition of apoptosis and ordered cell division. Since survivin is considered a bona fide cancer therapy target, these results strongly encourage future work to identify molecular decoys that specifically inhibit the nuclear export of survivin as novel therapeutics. In order to further dissect the regulation of nuclear transport and to efficiently identify transport inhibitors, cell-based assays are urgently required. Therefore, the cellular assay Systems developed in this work may not only serve to identify synthetic nuclear export and Import inhibitors but may also be applied in systematic RNAi-screening approaches to identify novel components of the transport machinery. In addition, the translocation based protease- and protein-interaction biosensors can be applied in various biological Systems, in particular to identify protein-protein interaction inhibitors of cancer relevant proteins. In summary, this work does not only underline the general significance of nucleo-cytoplasmic transport for cell biology, but also demonstrates its potential for the development of novel therapies against diseases like cancer and viral infections.
The analysis of doxorubicin-loaded poly(butyl cyanoacrylate) nanoparticles in in vitro glioma models
(2005)
The use of doxorubicin for the treatment of glioma tumours would be an important approach in the chemotherapy treatment since doxorubicin is a very effective neoplastic agent. However, one problem faced by the use of doxorubicin for the treatment of brain tumours is the fact that doxorubicin is a substrate of an efflux pump protein, P-glycoprotein (P-gp), which is located on the luminal side of the brain capillary endothelium and in many tumour cells, which acts pumping out of the cell such substrate, and blocking its transport into the cell. A strategy to enhance the doxorubicin delivery into the brain would be the use of nanoparticles. This work showed, that the treatment of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles decreased the viability of the three glioma cell lines, the GS-9L, the RG-2, and the F-98 cell lines significantly in comparison to doxorubicin in solution, indicating an improvement of the nanoparticles-bound doxorubicin transport into the cells. The modification of the nanoparticles surface with different surfactants may even enhance the delivery of the drug into the cells. Searching for an improvement of the doxorubicin internalization, the nanoparticles surface was modified using polysorbate 80, poloxamer 188 and poloxamine 908 surfactants. The poloxamer 188 and polaxamine 908 surfactant modified nanoparticles did not show a significant enhancement of the doxorubicin internalization. Contrary, the treatment of polysorbate 80 surfactant modified nanoparticles led in some cases to a significant decrease of cancer cell viability. The use of doxorubicin in the three glioma cell lines allowed the measurement of different responses towards doxorubicin treatment. The different responses were due to the entry of various amounts of doxorubicin into the glioma cells, which express the P-glycoprotein in their cellular membrane. A higher level of the P-gp expression correlated with a weaker response towards the doxorubicin treatment. The GS-9L cell line showed a significant higher level of P-gp expression than the F-98, and RG-2 cell lines, and consequently, the GS-9L cell line presented the highest resistance to doxorubicin with the highest viability values after doxorubicin treatment. Due to the fact that the transport of doxorubicin is governed by the activity of the P-gp in the studied glioma cells, the use of poloxamer 185 as a P-gp inhibitor resulted in an enhancement of the uptake as well as of the accumulation of doxorubicin into the cells. The effect of poloxamer 185 on the doxorubicin uptake was significant marked in the case of doxorubicin-resistance cells, as the GS-9L cell line. In some cases, the presence of the nanoparticles formulation showed also an influence on such uptake improvement. The use of a P-gp inhibitor in combination with chemotherapeutic agents leads to encouraging results. Because of the wide spectrum of substances acting as P-gp inhibitors, the exact inhibitory mechanisms remain still unclear. For instance in our results the evaluation of a described P-gp inhibitor, polysorbate 80 did not show an important improvement in the doxorubicin uptake in the P-gp-expressing cell line, GS-9L. On the other hand, the Polysorbate 80-Dox-PBCA nanoparticles formulation decreased in greater extend the viability of the glioma cells than the poloxamer185-Dox-PBCA nanoparticles. Although, the P-gp inhibition was undoubtedly higher in the presence of poloxamer 185, polysorbate 80 showed a main effect on the disruption of the cellular membrane, resulting in an important cellular viability decrease. It seems that poloxamer 185 presents a direct effect on the functionality of the P-gp protein, which would be of great importance in the sensitization of resistant cancer cells. The range of concentration of poloxamer 185 is very important to yield an inhibitory effect on the P-gp-mediated transport mechanism. The accumulation of Rhodamine-123 (Rho-123), a known P-gp substrate, increased in a range of concentration from 0.001 % to 0.01, whereas at 0.1 % poloxamer 185 the accumulation significantly decreased. A maximal Rho-123 accumulation was reached at 0.01 % poloxamer 185.
The results presented here strongly indicate that ubiquitination of the recombinant human alpha1 GlyR at the plasma membrane of Xenopus oocytes is involved in receptor internalisation and degradation. Ubiquitination of the human alpha1 GlyR has been demonstrated by radio-iodination of plasma membrane-boundalpha1 GlyRs, whose subunits differed in molecular weight by additional 7, 14 or 21 kDa, corresponding to the molecular weights of one, two and three conjugated ubiquitin molecules, respectively, and by co-isolation of the non-tagged human alpha1 GlyR through hexahistidyl-tagged ubiquitin. Ubiquitin conjugated GlyRs where prominent at the plasma membrane, but could be hardly detected in total cell homogenates, indicating that ubiquitination takes place exclusively at the plasma membrane. Ubiquitination of the alpha1 GlyR at the plasma membrane was no longer detectable when the ten lysine residues of the cytoplasmic loop between transmembrane segments M3 and M4 were replaced by arginines. Despite this proteolytic cleavage continued to take place at the same extent as with the wild type alpha1 GlyR, suggesting that removal of GlyRs from the plasma membrane and routing to lysosomes for degradation were not dependent on ubiquitination. Also replacing a tyrosine in position 339, which was speculated to be part of an additional endocytosis motif, did not lead to a significant reduction of cleavage of the GlyR alpha1 subunits. However, a mutant lacking both, ubiquitination sites and 339Y, was significantly less processed. These results may suggest that the GlyR alpha1 subunit harbors at least two endocytosis motifs, which may act independently to regulate the density of alpha1 GlyR. Apparently, each of the two signals may be capable of compensating entirely the loss of the other. Part two of this Dissertation demonstrates that the correct topology of the glycine receptor alpha1 subunit depends critically on six positively charged residues within a basic cluster, RFRRKRR, located in the large cytoplasmic loop following the C-terminal end of M3. Neutralization of one or more charges of this cluster, but not of other charged residues in the M3-M4 loop, led to an aberrant translocation into the endoplasmic reticulum lumen of the M3-M4 loop. However, when two of the three basic charges located in the ectodomain linking M2 and M3 were neutralized, in addition to two charges of the basic cluster, endoplasmic reticulum disposition of the M3-M4 loop was prevented. We conclude that a high density of basic residues C-terminal to M3 is required to compensate for the presence of positively charged residues in the M2-M3 ectodomain, which otherwise impair correct membrane integration of the M3 segment. Part three of this Dissertation describes my contribution (blue native PAGE analysis of metabolically labeled alpha7 and 5HT3A receptors and the examination of the glycosylation state of metabolically labeled alpha7 subunits) to a work on the limited assembly capacity of Xenopus oocytes for nicotinic alpha7 subunits. While 5HT3A subunits combined efficiently to pentamers, alpha7 subunits existed in various assembly states including trimers, tetramers, pentamers, and aggregates. Only alpha7 subunits that completed the assembly process to homopentamers acquired complex-type carbohydrates and appeared at the cell surface. We conclude that Xenopus oocytes have a limited capacity to guide the assembly of alpha7 subunits, but not 5HT3A subunits to homopentamers. Accordingly, ER retention of imperfectly assembled alpha7 subunits rather than inefficient routing of fully assembled alpha7 receptors to the cell surface limits surface expression levels of alpha7 nicotinic acetylcholine receptors. Part four of this Dissertation describes my contribution (the biochemical analysis of the human P2X2 and P2X6 subtypes) to studies on the quaternary structure of P2X receptors. Armaz Aschrafi, the main author of the paper showed that subsequent to isolation under non-denaturing conditions from Xenopus oocytes the His-rP2X2 protein migrated on blue native PAGE predominantly in an aggregated form. The only discrete protein band detectable could be assigned to homotrimers of the His-rP2X2 subunit. Because of the exceptional assembly-behaviour of the rP2X2 protein compared to the rP2X1, rP2X3, rP2X4 and rP2X5 proteins, its human orthologue was investigated in the same manner. In contrast to rP2X2 subunits, hP2X2 subunits migrated under virtually identical conditions in a single defined assembly state, which could be clearly assigned to a trimer. P2X6 subunits represent the sole P2X subtype that is unable to form functional homomeric receptors in Xenopus oocytes. The blue native PAGE analysis of metabolically labeled hP2X6 receptors and the examination of the glycosylation state revealed that hP2X6 subunits form tetramers and aggregates that are not exported to the plasma membrane of Xenopus oocytes.
Haematopoietic stem cells (HSCs) are regarded as the prime target for gene therapy of inherited and acquired disorders of the blood system, e.g. X-linked chronic granulomatous disease (X-CGD). The major reason for this is that HSCs posses the ability to self renew as well as the potential to differentiate into all lineage-specific cell types. However, the need to reach and to maintain sufficient therapeutic levels of genetically modified stem cells and their progeny after gene delivery still presents major challenges for current HSC gene therapy approaches. In particular, one of the main limitations for most genetic defects is the lack of a selective growth advantage of gene-modified cells after engraftment. In vitro and in vivo methods have been developed that focus on either positive or negative selection of HSCs. An artificial selection advantage can be conferred to transduced HSCs by incorporating a selection marker in addition to the therapeutic transgene. In the present study, two novel strategies for positive selection of murine gp91phox gene-modified haematopoietic stem cells were developed and tested, bearing in mind that with selective growth advantage, the possibility of uncontrolled proliferation arises. The first strategy to be investigated was based on the homeobox transcription factor HOXB4, which plays an important role in the control of haematopoietic stem cell proliferation and differentiation. Overexpression of a retroviral bicistronic construct containing the therapeutic gene gp91phox and HOXB4 in murine primary bone marrow cells led to a significant 3–4-fold expansion of transduced cells ex vivo. The numbers of transgene-expressing cells increased 2–3-fold after 2 weeks cultivation under cytokine stimulation. Furthermore, the clonogenic progenitor cell assay (CFU assay) demonstrated that the number of colony-forming cells had increased to levels 2-fold higher than those of mock-transduced cells after 1 week of culture, thereby augmenting the presence of a significant number of stem/progenitor cells in the selected cell population. However, in our experiments, HOXB4-overexpressing murine HSCs did not show any repopulating advantage in transplanted recipient mice over control construct-transduced HSCs. These results indicate that selective expansion of gp91phox gene-modified HSCs can be induced by the HOXB4 transcription factor ex vivo but not in vivo. This is possibly dependent on HOXB4 expression levels, which are too low in vivo to achieve selection. The second strategy made use of a chemically inducible dimerizer system consisting of the therapeutic gene gp91phox and a fusion protein, containing sequences from a growth factor receptor signalling domain (epidermal growth factor receptor, EGFR, or prolactin receptor, PrlR) and the drug binding protein FKBP12, as the selection cassette. This strategy aimed to allow inducible selection that could be easily switched off. The activity of these fusion proteins is controlled through the small molecular dimerizer AP20187. Transduction of BaF/3 cells with lentiviral vectors expressing the EGFR construct induced proliferation and led to complete selection within 18 days (99%). However, removing AP20187 could not turn off proliferation. This construct is, therefore, not suitable as a selection cassette for the expansion of gene-modified HSCs due to its oncogenic potential. Transduction of the construct containing the intracellular domain of PrlR caused significant selective expansion of AP20187-treated BaF/3 cells. Following expression in cells, the fusion protein, which lacks membrane-anchoring sequences, mainly localized to the cytoplasm. Evidence was found to indicate that activated STAT5 might be responsible for this effect. Upon expression of the prolactin construct, phosphorylation of STAT5 and its DNA-binding activity to a ß-casein promoter sequence was strongly increased. Importantly, the induced proliferation was reversible after removal of AP20187. Transduced Sca1+ bone marrow cells obtained from C57BL/6-CD45.1 mice could be expanded about 20–100-fold ex vivo in the presence of AP20187 and mSCF without losing progenitor cell features and the capability to contribute to all lineages of the haematopoietic system. To exclude oncogenic outgrowth of one single clone, the polyclonality of selected cells was proven by ligation-mediated PCR (LM-PCR) analysis. In mouse transplantation experiments, ex vivo-expanded cells repopulated the bone marrow of lethally irradiated mice suggesting that the ex vivo expansion took place at the level of haematopoietic stem and/or progenitor cells. Genomic gp91phox sequences were detected in the bone marrow, spleen and peripheral blood cells of transplanted animals, indicating that gp91phox-containing cells most likely contributed to the reconstitution of haematopoiesis in these mice.
Stem cells capable of self-renewal and differentiation into multiple tissues are important in medicine to reconstitute the hematopoietic system after myelo-ablative chemo- or radiotherapy. In the present situation, adult stem cells such as Mesenchymal stem cells (MSC) and Hematopoietic stem cells (HSC) are used for therapeutic purposes. For tissue regeneration and tissue constitution, engraftment of transplanted stem cells is a necessary feature. However, in many instances, the transplanted stem cells reach the tissues with low efficiency. Considering the three-step model of leukocyte extravasation by Springer et al, the rolling, adhesion and transmigration form the three major steps for the transplanted stem cells to enter the desired tissues. One of the molecular switches reported to be involved in these mechanisms are the Rho family GTPases. The present study investigates the role of Rho GTPases in adhesion and migration of stem and progenitor cells. Chemotactic and chemokinetic migration assays, transendothelial migration assays, migration of cells under shear stress, microinjection, retroviral and lentiviral gene transfer methods, oligonucleotide microarray analysis and pull down assays were employed in this study for the elucidation of Rho GTPase involvement in migration and adhesion of stem and progenitor cells. The transmigration assay used for the migration determination of the adherent cell type, MSC, was optimized for the efficient and effective assessment of the migrating cells. The involvement of Rho was found to be critical for stem and progenitor cell migration where inactivation of Rho by C2I-C3 transferase toxin and/or overexpression of C3 transferase cDNA increased the migration rate of Hematopoietic progenitor cells (HPC) and MSC. Moreover, modulation of Rho caused predictable cytoskeletal and morphological changes in MSC. Assessment of Rho GTPase involvement in the interacting partner, the endothelial cells during stem cell migration, revealed that active Rho expression induced E-selectin expression. The increased levels of E-selectin were functionally confirmed by the increased adhesion of progenitor cells (HPC) to the Human umbilical vein endothelial cell (HUVEC) layer. Moreover, inhibition of Rac in the migrating endothelial progenitor cells (eEPC) increased their adhesion to HUVEC correlating with the increased percentage expression of cell surface receptor, CD44 in Rac inactivated eEPC. In conclusion, this study shows that Rho GTPases control the adhesion and migration of stem and progenitor cells, HPC and MSC. Rho inhibition drives the cells to migrate in the blood vessels. The substantial increase in the level of active Rho in endothelial layer, manifested by the E-selectin surface expression assists the better adhesion of stem and progenitor cells to the endothelial layer. Serum factors and growth factors in the physiological system influence the Rho GTPase expression in both migrating stem cells and the barrier endothelial cells. Thus, specific modulation of Rho GTPases in the transplanted stem and progenitor cells could be an interesting tool to improve the migration and homing processes of stem cells for cellular therapy in future.
Systematisch verabreichte Chemotherapeutika sind oft uneffektiv bei der Behandlung von Krankheiten des zentralen Nervensystems (ZNS). Eine der Ursachen hierfür ist der unzureichende Arzneistoff-Transport ins Gehirn aufgrund der Blut-Hirn-Schranke. Eine der Strategien für den nicht-invasiven Wirkstoff-Transport ins Gehirn ist die Verwendung von Nanopartikeln. Polybutylcyanoacrylat-Nanopartikel, die mit Polysorbat 80 (Tween® 80) überzogen wurden, können die Blut-Hirn-Schranke passieren und somit Wirkstoffe ins Gehirn transportieren. Wird die Blut-Hirn-Schranke durch einen Hirntumor partiell beschädigt und hierdurch ihre Permeabilität am Ort des Tumors erhöht, können Nanopartikel den Tumor zusätzlich durch den sogenannten EPR-Effekt erreichen. Im ersten Teil der vorliegenden Arbeit wurde die Beladung der Nanopartikel durch Variation der Formulierungparameter mit dem Ziel optimiert, eine Formulierung mit höherer Wirksamkeit für die Therapie von Glioblastom-tragenden Ratten zu entwickeln. Außerdem wurde das Potential von Doxorubicin, das an mit „Stealth Agents“ überzogenen Polybutylcyanoacrylat-Nanopartikel gebunden war, für die Chemotherapie von Hirntumoren untersucht. Im zweiten Teil dieser Studie wurden die Gehirn- und Körperverteilung in gesunden und in Glioblastom-101/8-tragenden Ratten nach i.v.-Gabe von Poly(butyl-2-cyano[3- 14C]acrylat)-Nanopartikeln, die mit Polysorbat 80 beschichtet wurden, und solchen, die noch zusätzlich mit Doxorubicin geladen waren (DOX-14C-PBCA + PS), untersucht. Die Standardformulierung von Doxubicin-Polybutylcyanoacrylat-Nanopartikeln (DOX-NP) wurde durch anionische Polymerisierung von Butylcyanoacrylat in Anwesenheit von DOX hergestellt. Zusätzlich wurden unterschiedliche DOX-NP Formulierungen durch Veränderung der Herstellung produziert. Das therapeutische Potential der Formulierungen wurde in Ratten mit ins Gehirn transplantieren Glioblastom 101/8 untersucht. Neben Polysorbat 80 wurden Poloxamer 188 und Poloxamin 908 als Überzugsmaterial verwendet. Die Resultate ergaben, dass die mit Polysorbat 80 überzogene Standardformulierung am effektivsten war. Die höhere Wirksamkeit von DOX-NP+PS 80 könnte durch die Fähigkeit dieser Träger erklärt werden, den Wirkstoff während eines frühen Stadiums der Tumorentwicklung durch einen Rezeptor-vermittelten Mechanismus, der durch den PS 80-Überzug aktiviert wurde über die intakte Blut-Hirn-Schranke, zu transportieren. Unsere Ergebnisse zeigen auch, dass Poloxamer 188 und Poloxamin 908 den antitumoralen Effekt von DOX-PBCA beträchtlich verbessern. Der anti-tumorale Effekt dieser Formulierungen könnte möglicherweise dem EPR-Effekt zugeschrieben werden. Es ist bekannt, dass die tumorale Arzneistoff-Aufnahme durch den EPR-Effektes für lang-zirkulierende Wirkstoffträger ausgeprägter ist und so mehr Wirkstoff durch die Tumor-geschädigte Blut-Hirn-Schranke gelangt. Unbeschichtete Nanopartikel, Polysorbat 80-beschichtete Nanopartikel oder mit Doxorubicin beladene und mit Polysorbat 80 beschichtete Nanopartikel wurden in gesunden und Tumor-tragenden Ratten injiziert. Diese Nanopartikel-Präparationen zeigten einer unterschiedliche Korpenverteilung in den Ratten. Unbeschichtete Nanopartikel sammelten sich in den RES-Organen an. Mit PS 80 beschichtete NP reduzierten die Aufnahme der NP in Leber und Milz, während sich die Konzentration der NP in der Lunge erhöhte. Diese Beobachtungen deuten darauf hin, dass die Änderung der Oberflächeneigenschaften der NP durch das Tensid, zu einer Interaktion mit unterschiedlichen Opsoninen führt, welches die Aufnahme der NP von verschiedenen phagozitierenden Zellen erleichtert. Hingegen war die Aufnahme der mit DOX beladenen, PS 80-beschichteten Nanopartikel den unbeschichteten Partikel ähnlich. Im Vergleich mit gesunden Ratten und mit Tumor-tragenden Ratten hingegen war die Konzentration der NP im Gehirn von Tumor tragenden Ratten 10 Tage nach der Tumor-implantation signifikant höher. In Anwesenheit des Glioblastoms ist der Transport von NP in das Gehirn das Resultat verschiedener Faktoren: zusätzlich zur Fähigkeit von PS 80-Nanopartikeln, die Blut-Hirn-Schranke zu passieren, extravasieren diese Träger wegen des EPR Effekts über das durch den Tumor undichte Endothelium. Die Konzentration von PS 80 [14C]-PBCA NP war im Glioblastom signifikant höher als mit DOX [14C]-PBCA NP. Dieses Phänomen kann durch die unterschiedliche Mikroumgebung von zerebralem intra-tumoralen und intaktem Gehirngewebe erklärt werde. Insbesondere können sich die positive Ladung der tumoralen Regionen und die positive Ladung der DOX [14C]-PBCA NP negativ beeinflussen. Dennoch waren die Doxorubicin-Konzentration in Glioblastom ausreichend, einen therapeutischen Effekt zu ermöglichen.
The development of a drug product, beginning with the synthesis of the drug substance through approval for marketing, may take up to 15 years and a total amount of investment of up to half a billion Euro. After the discovery of a potential drug substance, many different investigations have to be performed: e.g. characterization of the physical-chemical properties, the pharmacological and toxicological profile and, especially relevant for this work, the development of the first dosage forms. After achieving these steps, first investigations in human studies can be carried out. After a positive assessment of the benefit to risk ratio, further investigations, such as food effects on the pharmacokinetics, multiple dosing studies and further studies on patients can be implemented. After successfully completing this second part the new drug product can be approved. With broader clinical experience it often becomes apparent that changes in relevant aspects of the formulation of the registered drug product e.g. excipients, concentration of the drug substance or excipient versus drug substance ratio, are necessary to optimise the therapy. This often leads to additional clinical investigations and a new registration, a procedure which is time and cost intensive. A possible way to reduce the financial and time investments, is to establish an appropriate in vivo- in vitro correlation (IVIVC). If it is possible to predict the in vivo performance of a drug product adequately with in vitro methods (dissolutions tests), it will no longer be necessary to perform additional clinical investigations. In this work, IVIVCs were investigated for three different drug substances and several different types of formulations.... ...Results of this work clearly show that successful IVIVCs can be achieved for the fasted state using biorelevant dissolution media. A prerequisite of achieving a good IVIVC is the availability of in vivo data of a reference product (i.v., oral solution or IR) tested within the same group of volunteers as the product of interest. Only with this procedure, one can obtain adequate IVIVCs for drug substances with high inter-individual variability of the plasma concentrations and with high first-pass metabolism. This work also shows that predictions of the in vivo behavior of a modified release dosage form after administration with a high fat meal are more difficult to obtain. This is mainly related to an absence of a medium, which could mimic the situation of the fed stomach adequately. Ensure plus®, which was chosen in this work, failed to simulate the fed stomach adequately in several cases; it suppressed the release of rosiglitazone from lipid formulations and led to rapid disruption of the HPMC-matrix of the 5-ISMN Geomatrix formulations. Future work should be directed towards optimization of the test media in the BioDis apparatus. This work clearly shows the inability of Ensure plus® to predict the in vivo performance of a drug under fed state conditions and indicates that alternative media must be developed. It is known that the pH of the stomach rises up to six after the intake of a meal. During the following hours the pH decreases until reaching the baseline value of approximately 1.8. One possibility of simulating the fed state stomach more precisely will be to divide the overall residence time into 4 different parts: 1. half a hour at pH 6 2. half a hour at pH 4 3. one hour at pH 3 4. two hours at pH 1.8 Another option is not only to modify the pH of the medium, but also to change its composition. During the decomposition of the food contents, the composition of the gastric juice changes, the ionic strength, the buffer capacity and the osmolarity rises, while the pH value decreases. A third possibility will be the addition of enzymes, mainly pepsin, lipases and amylases. Again, the quantity of the enzymes differs during the residence time of the food in the stomach. Highest quantities are expected in the first two hours after food intake and decreases in the remaining two hours. Another issue of this work was an assessment of the two dissolution apparatus, Paddle and BioDis. In general, the choice of the dissolution apparatus should be done primarily with respect to the solubility behavior of the drug substance. For high soluble drugs the USP apparatus II, Paddle, is sufficient (e.g. diltiazem or 5-ISMN). In cases of a poorly soluble drug (rosiglitazone), where the release strongly depends on the medium used, the USP apparatus III (BioDis) is favored, due to the advantage of simulating the GI-tract with a gradient of different dissolution media, each simulating one part of the GI-tract. In summary, the results of this work indicate that it is acurrently possible to predict fasted state behavior of a variety of controlled release products using in vitro tests. Prognoses was also made in terms of predicting food effects on the behavior of controlled release products, although it is clear that the media compositions will have to be revised to establish releiable predictive methods for the fed state.
In order to investigate the role of neuronal synchronization in perceptual grouping, a new method was developed to record selectively from multiple cortical sites of known functional specificity as determined by optical imaging of intrinsic signals. To this end, a matrix of closely spaced guide tubes was developed in cooperation with a company providing the essential manufacturing technique RMPD® (Rapid Micro Product Development). The matrix was embedded into a framework of hard and software that allowed for the mapping of each guide tube onto the cortical site an electrode would be led to if inserted into that guide tube. With these developments, it was possible to determine the functional layout of the cortex by optical imaging and subsequently perform targeted recordings with multiple electrodes in parallel. The method was tested for its accuracy and found to target the electrodes with a precision of 100 µm to the desired cortical locations. Using the developed technique, neuronal activity was recorded from area 18 of anesthetized cats. For stimulation, Gabor-patches in different geometrical configurations were placed over the recorded receptive fields merging into visual objects appropriate for testing the hypothesis of feature binding by synchrony. Synchronization strength was measured by the height of the cross-correlation centre peaks. All pairwise synchronizations were summarized in a correlation index which determined the mean difference of the correlation strengths between conditions in which recording sites should or should not fire in synchrony according to the binding hypothesis. The correlation index deviated significantly from zero for several of these configurations, further supporting the hypothesis that synchronization plays an important role in the process of perceptual grouping. Furthermore, direct evidence was found for the independence of the synchronization strength from the neuronal firing rate and for neurons that change dynamically the ensemble they participate in. In parallel to the experimental approach, mechanisms of oscillatory long range synchronization were studied by network simulations. To this end, a biologically plausible model was implemented using pyramidal and basket cells with Hodgkin-Huxley like conductances. Several columns were built from these cells and intra- and inter-columnar connections were mimicked from physiological data. When activated by independent Poisson spike trains, the columns showed oscillatory activity in the gamma frequency range. Correlation analysis revealed the tendency to locally synchronize the oscillations among the columns, but a rapid phase transition occurred with increasing cortical distance. This finding suggests that the present view of the inter-columnar connectivity does not fully explain oscillatory long range synchronization and predicts that other processes such as top-down influences are necessary for long range synchronization phenomena.
Acute myeloid leukemia (AML) is characterized by the accumulation of a large number of abnormal, immature blast cells. Recently, histone deacetylase inhibitors (HDIs) received considerable interest on the ground of their ability to overcome the differentiation block in these leukemic blasts regardless of the primary genetic alteration, an effect achieved either alone or in combination with differentiating agents, such as all-trans retinoic acid (t-RA). Valproic acid (VPA), a potent HDI, is now under clinical evaluation owing to its potent differentiation effect on transformed hematopoietic progenitor cells and leukemic blasts from AML patients. Conversely, in a clinical study by Bug et al., the favorable effects of the combination treatment with t-RA/VPA in advanced acute myeloid leukemia patients were reported to be most likely due to an enhancement of nonleukemic myelopoiesis and the suppression of malignant hematopoiesis rather than enforced differentiation of the leukemic cells. Based on the hypothesis that VPA influences normal hematopoiesis, the effect of chromatin modeling through VPA on HSCs was investigated with respect to differentiation, proliferation as well as self-renewal in the present study. It has been shown that valproic acid increases both proliferation and self-renewal of HSC. It accelerates cell cycle progression of HSC accompanied by a down-regulation of p21cip-1/waf-1. Furthermore, valproic acid inhibits GSK3B by phosphorylation on Ser9 accompanied by an activation of the Wnt signaling pathway as well as by an up-regulation of HoxB4, a target gene of Wnt signaling. Both are known to directly stimulate the proliferation of HSC and to expand the HSC pool. To sum up, valproic acid, a potent histone deacetylase inhibitor known to induce differentiation and/or apoptosis in leukemic blasts, stimulates the proliferation and self-renewal of hematopoietic stem cells. Therefore, the data reported in this study suggest to reconsider the role of histone deacetylase inhibitors from a differentiation inducer to a coadjuvant factor for increasing the response to conventional therapy in acute myeloid leukemia.
Drug target 5-lipoxygenase : a link between cellular enzyme regulation and molecular pharmacology
(2005)
Leukotriene (LT) sind bioaktive Lipidmediatoren, die in einer Vielzahl von Entzündungskrankheiten wie z.B. Asthma, Psoriasis, Arthritis oder allergische Rhinitis involviert sind. Des Weiteren spielen LT in der Pathogenese von Erkrankungen wie Krebs, Osteoarthritis oder Atherosklerose eine Rolle. Die 5-Lipoxygenase (5-LO) ist das Enzym, das für die Bildung von LT verantwortlich ist. Aufgrund der physiologischen Eigenschaften der LT, ist die Entwicklung von potentiellen Arzneistoffen, welche die 5-LO als Zielstruktur besitzen, von erheblichem Interesse. Die Aktivität der 5-LO wird in vitro durch Ca2+, ATP, Phosphatidylcholin und Lipidhydroperoxide (LOOH) und durch die p38-abhängige MK-2/3 5-LO bestimmt. Inhibitorstudien weisen darauf hin, dass der MEK1/2-Signalweg ebenfalls in vivo an der 5-LO Aktivierung beteiligt ist. Hauptziel dieser Arbeit war es zu untersuchen, welche Rolle der MEK1/2-Signalweg bei der Aktivierung der 5-LO besitzt und welchen Einfluss der 5-LO Aktivierungsweg auf die Wirksamkeit potentieller Inhibitoren hat. „In gel kinase“ und „In vitro kinase“ Untersuchungen zeigten, dass die 5-LO ein Substrat für die Extracellular signal-regulated kinase (ERK) und MK-2/3 darstellt. Der Zusatz von mehrfach ungesättigten Fettsäuren (UFA), wie AA oder Ölsäure, verstärkte den Phosphorylierungsgrad der 5-LO sowohl durch ERK1/2 als auch durch MK-2/3. Die genannten Kinasen sind demnach auch für die 5-LO Aktivierung durch natürliche Stimuli verantwortlich, die den zellulären Ca2+-Spiegel kaum beeinflussen. Daraus ist ersichtlich, dass die Phosphorylierung der 5-LO durch ERK1/2 und/oder MK-2/3 einen alternativen Aktivierungsmechanismus neben Ca2+ darstellt. Ursprünglich wurden Nonredox-5-LO-Inhibitoren als kompetitive Wirkstoffe entwickelt, die mit AA um die Bindung an die katalytische Domäne der 5-LO konkurrieren. Vertreter dieser Inhibitoren, wie ZM230487 und L-739,010, zeigen eine potente Hemmung der LT-Biosynthese in verschiedenen Testsystemen. Sie scheiterten jedoch in klinischen Studien. In dieser Arbeit konnten wir zeigen, dass die Wirksamkeit dieser Inhibitoren vom Aktivierungsweg der 5-LO abhängig ist. Verglichen mit 5-LO Aktivität, die durch den unphysiologischen Stimulus Ca2+-Ionophor induziert wird, erfordert die Hemmung zellstress-induzierter Aktivität eine 10- bis 100-fach höhere Konzentration der Nonredox-5-LO-Inhibitoren. Die nicht-phosphorylierbare 5-LO Mutante (Ser271Ala/Ser663Ala) war wesentlich sensitiver gegenüber Nonredox-Inhibitoren als der Wildtyp, wenn das Enzym durch 5-LO Kinasen aktiviert wurde. Somit zeigen diese Ergebnisse, dass, im Gegensatz zu Ca2+, die 5-LO Aktivierung mittels Phosphorylierung die Wirksamkeit der Nonredox-Inhibitoren deutlich verringert. Des Weiteren wurde das pharmakologische Profil des neuen 5-LO Inhibitors CJ-13,610 mittels verschiedener in vitro-Testsysteme charakterisiert. In intakten PMNL, die durch Ca2+-Ionophor stimuliert wurden, hemmte die Substanz die 5-LO Produktbildung mit einem IC50 von 70 nM. Durch Zugabe von exogener AA, wird die Wirkung vermindert und der IC50 des Inhibitors steigt an. Dies deutet auf eine kompetitive Wirkweise hin. Wie die bekannten Nonredox-Inhibitoren, verliert auch CJ-13,610 seine Wirkung bei erhöhtem zellulärem Peroxidspiegel. Der Inhibitor CJ-13,610 zeigt jedoch keine Abhängigkeit vom Aktivierungsweg der 5-LO. Grundsätzlich ist es also von fundamentaler Bedeutung bei der Entwicklung von neuen Arzneistoffen, die zellulären Zusammenhänge, insbesondere die Regulierung der Aktivität von Enzymen, zu kennen. Wie in dieser Arbeit gezeigt, hat die Phosphorylierung der 5-LO einen starken Einfluss auf die Regulation der 5-LO Aktivität und eine elementare Wirkung auf die Hemmung des Enzyms durch verschiedene Wirkstoffe.
Chemokines play a key role in the cellular infiltration of inflamed tissue. They are released by a wide variety of cell types during the initial phase of host response to injury, allergens, antigens, or invading microorganisms, and selectively attract leukocytes to inflammatory foci, inducing both migration and activation. Monocyte chemoattractant protein-1 (MCP-1), a member of the CC chemokine superfamily, functions in attracting monocytes, T lymphocytes, and basophils to sites of inflammation. MCP-1 is produced by monocytes, fibroblasts, vascular endothelial cells and smooth muscle cells in response to various stimuli such as tumour necrosis factor-a (TNF-a), interferon-g (IFN-g), and interleukin-1b (IL-1b). It also plays an important role in the pathogenesis of chronic inflammation, and overexpression of MCP-1 has been implicated in diseases including glomerulonephritis and rheumatoid arthritis. Oligonucleotide-directed triple helix formation offers a means to target specific sequences in DNA and interfere with gene expression at the transcriptional level. Triple helix-forming oligonucleotides (TFOs) bind to homopurine/homopyrimidine sequences, forming a stable, sequence-specific complex with the duplex DNA. Purine-rich sequences are frequent in gene regulatory regions and TFOs directed to promoter sequences have been shown to prevent binding of transcription factors and inhibit transcription initiation and elongation. Exogenous TFOs that bind homopurine/ homopyrimidine DNA sequences and form triple-helices can be rationally designed, while the intracellular delivery of single-stranded RNA TFOs has not been studied in detail before. In this study, expression vectors were constructed which directed transcription of either a 19 nt triplex-forming pyrimidine CU-TFO sequence targeting the human MCP-1 or two different 19 nt GU- or CA-control sequences, respectively, together with the vector encoded hygromycin resistance mRNA as one fusion transcript. HEK 293 cells were stable transfected with these vectors and several TFO and control cell lines were generated. Functional relevant triplex formation of a TFO with a corresponding 19 bp GC-rich AP-1/SP-1 site of the human MCP-1 promoter was shown. Binding of synthetic 19 nt CUTFO to the MCP-1 promoter duplex was verified by triplex blotting at pH 6.7. Underlining binding specificity, control sequences, including the GU- and CA-sequence, a TFO containing one single mismatch and a MCP-1 promoter duplex containing two mismatches, did not participate in triplex formation. Establishing a magnetic capture technique with streptavidin microbeads it was verified that at pH 7.0 the 19 nt TFO embedded in a 1.1 kb fusion transcript binds to a plasmid encoded MCP-1 promoter target duplex three times stronger than the controls. Finally, cell culture experiments revealed 76 ± 10.2% inhibition of MCP-1 protein secretion in TNF-a stimulated CU-TFO harboring cell lines and up to 88% after TNF-a and IFN-g costimulation in comparison to controls. Expression of interleukin-8 (IL-8) as one TNF-a inducible control gene was not affected by CU-TFO, demonstrating both highly specific and effective chemokine gene repression. Furthermore, another chemokine target, regulated upon activation normal T cell expressed and secreted (RANTES), which plays an essential role in inflammation by recruiting T lymphocytes, macrophages and eosinophils to inflammatory sites, was analysed using the triplex approach. A 28 nt TFO was designed targeting the murine RANTES gene promoter, and gel mobility shift assays demonstrated that the phosphodiester TFO formed a sequencespecific triplex with the double-stranded target DNA with a Kd of 2.5 x 10-7 M. It was analysed whether RANTES expression could be inhibited at the transcriptional level testing the TFO in two different cell lines, T helper-1 lymphocytes and brain microvascular endothelial cells (bend3 cells). Although there was a sequence-specific binding of the TFO detectable in the gel shift assays, there was no inhibitory effect of the exogenously added and phosphorothioate stabilised TFO on endogenous RANTES gene expression visible. Additionally, the small interfering RNA (siRNA) approach was tested as another strategy to inhibit expression of the pro-inflammatory chemokines MCP-1 and RANTES. Two different methods were pursuit, describing transient transfection with vector derived and synthetic siRNA. The vector pSUPER containing the siRNA coding sequence was used to suppress endogenous MCP-1 in HEK 293 cells. An empty vector without RNA sequence served as a control. Inhibition due to the siRNA was measured in stimulated and unstimulated cells. In TNF-a stimulated cells MCP-1 protein synthesis was decreased by 35 ± 11% after siRNA transfection. Using a synthetic double-stranded siRNA, the TNF-a induced MCP-1 protein secretion could be successfully inhibited about 62.3 ± 10.3% in HEK 293 cells, indicating that the siRNA is functional in these cells to suppress chemokine expression. The siRNA approach targeting murine RANTES in Th1 cells and b-end3 cells revealed no inhibition of endogenous gene expression. Gene therapy approaches rely on efficient transfer of genes to the desired target cells. A wide variety of viral and nonviral vectors have been developed and evaluated for their efficiency of transduction, sustained expression of the transgene, and safety. Among them, lentiviruses have been widely used for gene therapy applications. In order to improve the delivery of TFOs or siRNAs into the target cells, cloning of the lentiviral transfer vector SEW, the production of lentiviral particles by transient transfection were performed with the aim to generate lentiviral vector-derived TFOs in further experiments. Here, Th1 cells were transduced with infectious lentiviral particles and transduction efficacy was measured. Transduction efficacy higher than 82% could be achieved using the lentiviral vector SEW, opening optimal possibilities for the TFO or siRNA approach.