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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 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.
Many highly active antitumour agents are currently not employable for the systemic chemotherapy of brain tumours since their entrance into the brain is blocked by the BBB. Obviously, the development of a strategy allowing effective delivery of these agents across the BBB would enormously extend the potential of the systemic chemotherapy. Chemotherapy of rat glioblastoma using nanoparticle-bound doxorubicin Doxorubicin bound to polysorbate-coated nanoparticles had been previously shown to significantly enhance survival in the orthotopic rat 101/8 glioblastoma model. The objective of this study was to investigate the therapeutic effects of this formulation by morphometric, histological and immunohistological methods. The 101/8 glioblastoma was implanted intracranially into the male Wistar rats. The animals were randomly divided into 3 groups; one group served as untreated control (n = 20). The second group received doxorubicin in solution (Dox-sol, n = 18), and the third group received doxorubicin bound to PBCA nanoparticles coated with PS 80 (Dox-NP + PS 80, n = 18). The treatment regimen was 3 × 1.5 mg/kg on days 2, 5, and 8 after tumor implantation. The formulations were injected into the tail vein. The untreated control animals were sacrificed on days 6, 8, 10, 12, and 14 after the implantation. The animals that had received chemotherapy were sacrificed on day 10, 14 and 18 after the implantation. The brains were investigated by morphometrical, histochemical, and immunohistochemical methods such as the measurement of the tumor size, proliferation of tumor cells, vessel density, expression of glial fibrillary acidic protein (GFAP), expression of vascular endothelial growth factor (VEGF), incidence and dimension of necrosis, and microvascular proliferation. Tumours showed signs of malignancy including invasion to brain tissue and brisk mitotic activity. The tumor proliferation remained stable at high levels throughout the host survival time. Overall, the tumor showed a reproducible growth pattern and temporal development that is comparable to human glioblastoma. Furthermore, the 101/8 glioblastoma had infiltrated diffusely the surrounding host brain at the edge of the solid tumor mass showed no signs of encapsulation. Thus the 101/8 glioblastoma fulfills the most criteria for an adequate glioma model and can be qualified as a reliable model. ...
The NS5B protein of the hepatitis C virus (HCV) is a RNA-dependent RNA polymerase, which is the key enzyme for viral replication. It is recognized as one of the promising targets for antiviral intervention within the new HCV treatment approach of direct-acting antivirals (DAA). However, several of the known non-nucleoside HCV polymerase inhibitors (NNIs) identified by screening approaches show limitations in the coverage of all six major HCV genotypes (GT). Genotypic profiling therefore has to be implemented early in the screening cascade to discover new broadly active NNIs. This implies knowledge of the specific individual biochemical properties of polymerases from all GTs which is to date limited to GT 1 only. The work submitted here gives a comprehensive overview of the biochemical properties of HCV polymerases derived from all major GTs 1 - 6. Biochemical analysis of polymerases from 38 individual sequences revealed that the optima for monovalent cations, pH and temperature were similar between the GTs, whereas significant differences concerning concentration of the preferred cofactor Mg2+ were identified. Implementing the optimal requirements for the polymerases from each individual GT led to significant improvements in their enzymatic activities. However, the specific activity was distributed unequally across the GTs and could be ranked in the following descending order: 1b, 6a > 2a, 3a, 4a, 5a > 1a. Furthermore, the optimized assay conditions for GT profiling were confirmed by testing the inhibitory activity of four known prototype NNIs, each addressing one of the four NNI binding sites. Additionally, a novel NNI chemotype - identified by screening - is described, the substituted N-phenyl-benzenesulphonamides (SPBS). This inhibitor class showed reversible inhibition of NS5B from HCV 1b Con1 with IC50 values up to 39 nM. Based on the decreased inhibitory activity against a recombinant NS5B protein carrying the mutation L419M, it was assumed that the SPBS inhibitors bound to the thumb site II as it has been described for the carboxy thiophene inhibitors. The postulated binding site was consequently confirmed by analysing a provided co-crystal structure of NS5B in complex with a SPBS analogue. Notably, the two SPBS analogues SPBS-1 and SPBS-2 reported here revealed significant differences in addressing the NH-group of the main chain Y477 by hydrogen-bonds, watermediated or directly, which provoked a shift of the carboxyphenyl group of the inhibitors towards the H475 position for the water-mediated binding mode. Interestingly, the differences observed in the binding mode led to a different cross resistance profile at positions M423 and I482. Using the previously optimized biochemical primer-dependent transcription assay, inhibitory activity of the SPBS could be demonstrated against polymerases from HCV GTs 1a and 1b whereas the inhibitor class failed to inhibit any of the non-GT 1 polymerases. Furthermore, initial antiviral activity for SPBS was demonstrated against the subgenomic replicons of HCV GTs 1a and 1b, respectively, and no considerable cytotoxic potential against a panel of ten different cell types. Finally, concerning a possible future treatment without PEG-IFN α or ribavirin, the SPBS analogues were found to display additive to synergistic effects in combination with the benzothiadiazine, the benzofuran and the indole - representative inhibitors for the binding sites palm I, palm II and thumb I, repectively - in the biochemical assay. Within the same binding site as the SPBS, the reference compound hydroxydihydropyranone displayed additive interactions only with the benzothiadiazine (palm I) in the biochemical assay as well as in cell culture. Hence it could be concluded that, having characterized one individual NNI, no universal predication is possible concerning the combinatory behaviour of NNIs binding to the same binding site. As synergistic, antagonistic or additive interactions are inhibitor-dependent (not binding sitedependent) each novel NNI has to be characterized individually in one-to-one combinations.
Natural killer (NK) cells are white blood lymphocytes of the innate immune system that have diverse biological functions, including recognition and destruction of certain microbial infections and neoplasms [1]. NK cells comprise ~ 10% of all circulating lymphocytes and are also found in peripheral tissues including the liver, peritoneal cavity and placenta. Resting NK cells circulate in the blood, but, following activation by cytokines, they are capable of extravasation and infiltration into most tissues that contain pathogen-infected or malignant cells [2-5]. NK cells discriminate between normal and abnormal cells (infected or transformed) through engagement and dynamic integration of multiple signaling pathways, which are initiated by germline-encoded receptors [6-8]. Healthy cells are protected from NK cell-mediated lysis by expression of major histocompatibility complex (MHC) class I ligands for NK cell inhibitory receptors [6, 9]. The MHC is a group of highly polymorphic glycoproteins that are expressed by every nucleated cell of vertebrates, and that are encoded by the MHC gene cluster. The human MHC molecules are termed human leucocyte antigen (HLA)-A, B and C molecules. Every NK cell expresses at least one inhibitory receptor that recognizes a self-MHC class I molecule. So, normal cells that express MHC class I molecules are protected from self-NK cells, but transformed or infected cells that have down-regulated MHC class I expression are attacked by NK cells [10]. There are 2 distinct subsets of human NK cells identified mainly by cell surface density of CD56. The majority (approximately 90%) of human NK cells are CD56dimCD16bright and express high levels of FcγRIII (CD16), whereas a minority (approximately 10%) are CD56brightCD16dim/- [11]. Resting CD56dim NK cells are more cytotoxic against NK-sensitive targets than CD56bright NK cells [12]. However, after activation with interleukin (IL)-2 or IL-12, CD56bright cells exhibit similar or enhanced cytotoxicity against NK targets compared to CD56dim cells [12-14]. The functions of NK cells are regulated by a balance of signals (Fig. 1.1). These are transmitted by inhibitory receptors, which bind MHC class I molecules, and activating receptors, which bind ligands on tumors and virus-infected cells [15]. These receptors are completely encoded in the genome, rather than being generated by somatic recombinations, like T- and B-cell receptors.