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Das zeitgleiche Auftreten eines ischämischen Schlaganfalls sowie eines Takotsubo-Syndroms (TTS) scheint eine relevante, bisher nicht ausreichend verstandene klinische Konstellation zu sein. Die Pathologien können als über die Hirn-Herz-Achse gekoppelt verstanden werden, in die die Blut-Hirn-Schranke (BHS) als funktionale Komponente integriert ist. Das klinisch-neurologische Outcome dieses Patient:innen-Kollektivs scheint signifikant schlechter zu sein als nach solitärem ischämischen Insult. Es wurde hypothetisiert, dass die BHS in besonderem Maße kompromittiert sein könnte. Das vorwiegend weibliche, postmenopausale Patient:innenkollektiv präsentierte laborchemisch elevierte Katecholaminspiegel sowie Entzündungsparameter. Diese Konditionen wurden unter Sauerstoff-Glucose-Entzug (OGD) in vitro simuliert und resultierende Alterationen eines etablierten BHS-Modells aus murinen cEND-Zellen der cerebralen Mikrozirkulation untersucht. Die Evaluation der BHS-Integrität erfolgte anhand von spezifischen Junktionsproteinen sowie Integrinuntereinheiten. Alle Versuche wurden parallel unter Östrogen-Applikation (E2) durchgeführt, um die mögliche BHS-Protektion durch das weibliche Sexualhormon zu untersuchen. Die getrennte Applikation von Katecholaminen (KAT) sowie Entzündungsmediatoren (INF) führte gegenüber der simultanen Applikation zu einem geringeren BHS-Schaden. Dieser erschien zeitgebunden, wobei sich das Ausmaß gewissermaßen proportional zur Einwirkdauer verhielt. Auswirkungen von OGD sowie einer Reoxygenierung, im Sinne einer simulierten Reperfusion, potenzierten sich mit den Effekten von KAT/INF. Überwiegend kompromittierten OGD und KAT/INF die BHS-Integrität, wobei nach Reoxygenierung eine „Erholung“ oder ein „Reperfusionsschaden“ vorlag. Eine Protektion durch E2 war morphologisch nachweisbar, speziell gegenüber OGD, KAT/INF sowie einem „Reperfusionsschaden“. Auf Ebene der Gen- sowie Proteinexpression konnte dies nicht gezeigt werden. Die Homöostase des ZNS würde in vivo beeinträchtigt, Katecholamine sowie Entzündungsmediatoren könnten ungehindert das bereits durch die Ischämie geschädigte neuronale Gewebe erreichen. Insgesamt trägt diese Arbeit zu einem Verständnis der molekularen BHS-Veränderungen im Kontext des zeitgleichen Auftretens von TTS und einem ischämischem Insult bei. Es wurde eine experimentelle Grundlage geschaffen, um zukünftig pathogenetische Hintergründe weiter erforschen zu können. Darauf aufbauend könnten, nach weiterer in vitro- sowie in vivo-Forschung, klinische Therapiekonzepte optimiert werden.
Tumore von Kopf und Hals gehen weiterhin mit einer schlechten Prognose einher. Im Rahmen einer operativen Therapie tritt Wundsekret (WS) aus, welches der Wundheilung dient. Dieses kann in Kontakt mit Tumorzellen bzw. Resttumor in der Wunde kommen.
Im Rahmen der vorliegenden Arbeit wurde die Frage nach dem Einfluss von Wundsekret auf Zellvermehrung, Chemoresistenzentwicklung, den Zellzyklus und die Induktion einer Epithelial-mesenchymalen Transition (EMT) in Tumorzellen von Kopf und Hals gestellt.
Hierfür wurde das WS von Tag1 und das WS von Tag 2 im Dotblot auf seine Zytokinzusammensetzung analysiert. Zwei Tumorzelllinien von Kopf und Hals, FaDu und HlaC78, wurden mit WSTag1 und WSTag2 behandelt und untersucht, welche Effekte das WS auf die Zellen hat.
Verwendet wurden ein Proliferationsassay, eine Zellzyklusuntersuchung und Apoptosetestung mittels FACS, eine PCR, ein Spheroidmodell und die Lichtmikroskopie.
Im WS wurden erhöhte Konzentrationen verschiedener Zytokine, insbesondere von IL-6, nachgewiesen. Gezeigt werden konnte eine gesteigerte Proliferationsrate der Tumorzellen unter WS-Behandlung, jedoch keine veränderte Verteilung der Zellzyklusphasen. In HlaC78-Zellen konnte eine vermehrte Vitalität nach Cisplatinbehandlung nachgewiesen werden. In beiden Tumorzelllinien fand sich eine vermehrte Exprimierung von Snail 1, Snail 2 und Vimentin. E-Cadherin wurde vermindert exprimiert. Twist und N-Cadherin wiesen keine Veränderungen auf. Es zeigte sich eine vermehrte Migration der Tumorzellen in die Umgebung. Die Zellen wiesen nach Behandlung mit WS vermehrt mesenchymale Zeichen auf. Es konnte kein Unterschied der Auswirkungen einer Behandlung mit WSTag1 im Vergleich zu einer Behandlung mit WSTag2 festgestellt werden.
Insgesamt scheint WS in Tumorzellen von Kopf und Hals einen EMT-artigen Prozess in Gang zu setzen, also eine partial EMT (pEMT).
Als mögliche Auslöser dieser Veränderungen kommen die im WS nachgewiesenen Zytokine und v. a. IL-6 in Frage.
Vergleich der Bakterienlast in vivo und Wachstumskinetik in vitro hyperletaler Meningokokkentypen
(2020)
Die invasive Meningokokkenerkrankung stellt weltweit mit einer Letalität von 5-10% trotz antibiotischer Therapie eine Herausforderung dar. Ein spezifisches Virulenzgen, welches die Schwere der Meningokokkenerkrankung bestimmt, konnte bisher nicht definiert werden. Vorangegangene Studien zeigen eine Korrelation der Letalität mit der Bakterienlast, Unterschiede bezüglich der Letalität je nach Serogruppe, eine erhöhte Letalität bei Infektionen mit sogenannten hyperletalen Feintypen (bisher nicht veröffentlichte Daten des NRZMHi) sowie einen Unterschied in der maximal in Flüssigkultur erreichten Konzentration der Bakterien zwischen invasiven Stämmen und Trägerstämmen.
In dieser Arbeit wurden mögliche Gründe für die Hyperletalität bestimmter Meningokokkentypen experimentell untersucht. Insbesondere wird die Frage analysiert, ob die hyperletalen Meningokokkentypen mit einer höheren bakteriellen Last im Blut assoziiert sind und ob sie andere Wachstumscharakteristiken im Vergleich zu ihren Kontrollstämmen in vitro zeigen.
Hierzu erfolgte mittels quantitativer Echtzeit-Polymerase-Kettenreaktion die Bestimmung der bakteriellen Last in 62 Blutproben von Patienten mit bestätigter invasiver Meningokokkenerkrankung über den Nachweis des ctrA-Gens. Darunter waren elf Proben des hyperletalen Feintyps B:P1.7-2,4:F1-5 und fünf Proben des hyperletalen Feintyps C:P1.5,2:F3-3.
Die Wachstumsversuche wurden mit 30 zufällig gewählten Stämmen der hyperletalen Feintypen B:P1.7-2,4:F1-5, C:P1.5-1,10-8:F3-6 und C:P1.5,2:F3-3 mit ihren jeweiligen nach Alter und Geschlecht abgeglichenen nicht zu der Gruppe der hyperletalen Feintypen gehörenden Kontrollstämmen in dem Medium PPM+ durchgeführt.
Die Wachstumsgeschwindigkeit μ sowie die Kapazität A (maximale Konzentrationszunahme als Logarithmus der gemessenen OD im Verhältnis zur Ausgangsdichte ODT0) wurden durch nicht-lineare Regression anhand der modifizierten Gompertz-Funktion ermittelt. Die Messung der optischen Dichte erfolgte alle 30 Minuten über 16 Stunden bei 620nm durch das Gerät TECAN Infinite 200 Pro (Tecan Group Ltd., Männedorf / Schweiz). Die Methode wurde anhand einer publizierten Studie zwischen Trägerstämmen und invasiven Stämmen (Schoen et al., 2014) validiert und bestätigte einen marginalen Unterschied in der optischen Dichte (p=0,057, Wilcoxon-Test) zwischen den Gruppen. Es zeigte sich kein Unterschied in der Wachstumsgeschwindigkeit.
Aus den Ergebnissen dieser Arbeit können drei wesentliche Schlussfolgerungen gezogen werden:
1.) Die Bakterienlast in dieser Stichprobe ist, entgegen der Literatur, nicht abhängig von der Serogruppe und dem Feintyp, jedoch von der Krankheitsmanifestation.
2.) Die Kapazität A ist in der Gruppe der „hyperletalen“ Typen im Vergleich zu den Kontrollstämmen möglicherweise höher.
3.) Größere Stichproben (Nativmaterial, Stämme) sind erforderlich, um die Beobachtungen dieser Studie zu bestätigen.
Molecular Effects of Polyphenols in Experimental Type 2 Diabetes Mellitus and Metabolic Syndrome
(2019)
The growing prevalence of type 2 diabetes mellitus (T2DM) demands novel therapeutic and adjuvant strategies. Polyphenols (PPs) are plant secondary metabolites. Epidemiological studies demonstrate an inverse relationship between their increased intake and the risk of development of T2DM and cardiovascular complications. However, the PPs’ mechanism of action remains largely unknown. The present work aimed to expand knowledge regarding the effects of PPs on diabetes relevant molecular targets.
Pycnogenol® (PYC) is a standardized pine bark extract which consists of oligomeric and monomeric PPs. Its anti-diabetic effects have been demonstrated in clinical trials. As a part of a human study involving 20 healthy volunteers, the extract’s effects on dipeptidyl peptidase IV (DPP IV) were investigated. This protease terminates the insulin secretagogue action of incretins. Its inhibition is a promising strategy in T2DM treatment. This study uncovered that PYC-intake of 100 mg daily over 14 days statistically significantly reduced DPP IV serum concentrations by 8.2 % (n= 38, p= 0.032). Contrary to expectations, this decrease was not paralleled by a reduction in the serum DPP IV enzymatic activity. To the best of our knowledge, the present study was the first investigating the effects of PPs on DPP IV serum concentrations and activities in humans. The finding that PYC is capable of reducing DPP IV serum concentrations might be important with regard to diabetes, where DPP IV levels are increased.
Screenings for PPs’ in vitro effects on DPP IV activity were performed employing a purified enzyme. The effects of tested PPs (among which PYC ingredients) at a physiologically relevant concentration of 5 µM were weak (< 10 %) and too small compared to the reference compound sitagliptin, and thus not likely to be clinically relevant. This result is in discordance with some published data, but consistent with the outcome from the present human study. In addition, fluorescence interactions with the experimental setup were registered: under certain conditions urolithin B exhibited an autofluorescence which might mask eventual inhibitory activity. Quercetin quenched the fluorescence slightly which might contribute to false positive results. No statistically significant effects of selected constituents and metabolites of PYC on the total DPP IV protein expression were observed in 3T3-L1 adipocytes. Thus, the lower DPP IV in vivo concentrations after intake of PYC cannot be explained with down-regulation of the DPP IV expression in adipocytes.
Akt kinase is responsible for the transmission of insulin signals and its dysregulation is related to insulin resistance and plays an important role in development of cardiovascular complications in T2DM. Thus, the modulation of the phosphorylation status of endothelial Akt-kinase, respectively its activity, might be a promising strategy in the management of these pathologies. This work aimed to uncover the effects of PPs from different structural subclasses on Akt-phosphorylation (pAkt) in endothelial cells (Ea.hy926). Short-term effects (5 – 30 min) were investigated at a concentration of 10 µM. In a pilot study two model PPs induced a moderate, but reproducible inhibition of pAkt Ser473 of 52.37 ± 21.01 % (quercetin; p= 0.006, n= 3) and 37.79 ± 7.14 % (resveratrol; p= 0.021, n= 4) compared to the negative control. A primary screening with Western blot analysis investigated the effects of eight compounds from different subclasses on pAkt Ser473 and Thr308 to reveal whether the observed inhibition PPs a group effect or specific to certain compounds. In addition to resveratrol and quercetin, statistically significant inhibitions of pAkt Ser473 were induced by luteolin (29.96 ± 11.06 %, p< 0.01, n= 6) and apigenin (22.57 ± 10.30 %, p< 0.01, n= 6). In contrast, genistein, 3,4,5-trimethoxystilbene, taxifolin and (+)-catechin caused no inhibition. A strong positive and statistically significant correlation between the mean inhibitory effects of the tested PPs on both Akt-residues Ser473 and Thr308 (r= 0.9478, p= 0.0003) was determined. A comprehensive secondary screening via ELISA involving 44 compounds from nine structural groups quantified the effects of PPs on pAkt Ser473 to uncover potential structure-activity features. The most potent inhibitors were luteolin (44.31 ± 17.95 %), quercetin (35.71 ± 8.33 %), urolithin A (35.28 ± 11.80 %), apigenin (31.79 ± 6.16 %), fisetin (28.09 ± 9.09 %), and resveratrol (26.04 ± 5.58 %). These effects were statistically significant (p< 0.01, n= 3 to 6). Further lead structure optimization might be based on the fact that the effects of luteolin and resveratrol also differed statistically significantly from each other (p= 0.008).
To the best of our knowledge, the present study is the first to compare quantitatively the short term effects of PPs from different subclasses on pAkt in endothelial cells. Basic structure-activity relationships revealed that for flavones and flavonols the presence of a C2=C3 double bond (ring C) was essential for inhibitory activity and hydroxylation on the m- and p- positions in the ring B contributed to it. For stilbenoids, three free OH-groups appeared to be optimal. The comparison of the inhibitory potentials of ellagic acid and its microbial metabolites showed that urolithin A was statistically significantly more effective than its progenitor compound. Despite their structural similarities, the only active compound among all urolithins tested was urolithin A, hydroxylated at the C3 and C8 positions. This suggested a specific effect for urolithin A. Based on the common structural determinants and molecular geometry of the most active PPs a pharmacophore model regarding Akt-inhibition was proposed.
In summary, the effects of a wide variety of PPs from diverse structural subclasses on the in vitro phosphorylation of endothelial Akt were quantitatively analyzed for the first time, the effects of previously undescribed compounds were determined and structure activity relationships were elucidated. The inhibitory potential of individual PPs might be beneficial in cases of sustained over-activation of Akt-kinase and its substrates such as S6 kinase as reported for certain T2DM-related pathological states, such as insulin resistance, endothelial dysfunction, excessive angiogenesis, vascular calcification, and insulin triggered DNA-damage. The results of the present work suggest potential molecular mechanisms of action of PP involving Akt-inhibition and DPP IV-down-regulation and thus contribute to the understanding of anti-diabetic effects of these compounds on the molecular level.
The current treatment strategies for diseases are assessed on the basis of diagnosed phenotypic changes due to an accumulation of asymptomatic events in physiological processes. Since a diagnosis can only be established at advanced stages of the disease, mainly due to insufficient early detection possibilities of physiological disorders, doctors are forced to treat diseases rather than prevent them. Therefore, it is desirable to link future therapeutic interventions to the early detection of physiological changes. So-called sensor-effector systems are designed to recognise disease-specific biomarkers and coordinate the production and delivery of therapeutic factors in an autonomous and automated manner. Such approaches and their development are being researched and promoted by the discipline of synthetic biology, among others.
Against this background, this paper focuses on the in vitro design of cytokine-neutralizing sensor-effector cells designed for the potential treatment of recurrent autoimmune diseases, especially rheumatoid arthritis.
The precise control of inducible gene expression was successfully generated in human cells. At first, a NF-κB-dependent promoter was developed, based on HIV-1 derived DNA-binding motives. The activation of this triggerable promoter was investigated using several inducers including the physiologically important NF-κB inducers tumor necrosis factor alpha (TNFα) and interleukin 1 beta (IL-1β). The activation strength of the NF-κB-triggered promoter was doubled by integrating a non-coding RNA. The latter combined expressed RNA structures, which mimic DNA by double stranded RNAs and have been demonstrated to bind to p50 or p65 by previous publications. The sensitivity was investigated for TNFα and IL-1β. The detection limit and the EC50 values were in in the lower picomolar range. Besides the sensitivity, the reversibility and dynamic of the inducible system were characterized. Hereby a close correlation between pulse times and expression profile was shown.
The optimized NF-κB-dependent promoter was then coupled to established TNFα- and IL-1-blocking biologicals to develop sensor-effector systems with anti-inflammatory activity, and thus potential use against autoimmune diseases such as rheumatoid arthritis. The biologicals were differentiated between ligand-blocking and receptor-blocking biologicals and different variants were selected: Adalimumab, etanercept and anakinra. The non-coding RNA improved again the activation strength of NF-κB-dependent expressed biologicals, indicating its universal benefit. Furthermore, it was shown that the TNFα-induced expression of NF-κB-regulated TNFα-blocking biologics led to an extracellular negative feedback loop. Interestingly, the integration of the non-coding RNA and this negative feedback loop has increased the dynamics and reversibility of the NF-κB-regulated gene expression. The controllability of drug release can also be extended by the use of inhibitors of classical NF-κB signalling such as TPCA-1. The efficacy of the expressed biologicals was detected through neutralization of the cytokines using different experiments. For future in vivo trials, first alginate encapsulations of the cells were performed. Furthermore, the activation of NF-κB-dependent promoter was demonstrated using co-cultures with human plasma samples or using synovial liquids.
With this generated sensor-effector system we have developed self-adjusting cytokine neutralizer cells as a closed-loop delivery system for anit-inflammatory biologics.
The main function of the small intestine is the absorption of essential nutrients, water and vitamins. Moreover, it constitutes a barrier protecting us from toxic xenobiotics and pathogens. For a better understanding of these processes, the development of intestinal in vitro models is of great interest to the study of pharmacological and pathological issues such as transport mechanisms and barrier function. Depending on the scientific questions, models of different complexity can be applied.
In vitro Transwell® systems based on a porous PET-membrane enable the standardized study of transport mechanisms across the intestinal barrier as well as the investigation of the influence of target substances on barrier integrity. However, this artificial setup reflects only limited aspects of the physiology of the native small intestine and can pose an additional physical barrier. Hence, the applications of this model for tissue engineering are limited.
Previously, tissue models based on a biological decellularized scaffold derived from porcine gut tissue were demonstrated to be a good alternative to the commonly used Transwell® system. This study showed that preserved biological extracellular matrix components like collagen and elastin provide a natural environment for the epithelial cells, promoting cell adhesion and growth. Intestinal epithelial cells such as Caco-2 cultured on such a scaffold showed a confluent, tight monolayer on the apical surface. Additionally, myofibroblasts were able to migrate into the scaffold supporting intestinal barrier formation.
In this thesis, dendritic cells were additionally introduced to this model mimicking an important component of the immune system. This co-culture model was then successfully proven to be suitable for the screening of particle formulations developed as delivery system for cancer antigens in peroral vaccination studies. In particular, nanoparticles based on PLGA, PEG-PAGE-PLGA, Mannose-PEG-PAGE-PLGA and Chitosan were tested. Uptake studies revealed only slight differences in the transcellular transport rate among the different particles. Dendritic cells were shown to phagocytose the particles after they have passed the intestinal barrier. The particles demonstrated to be an effective carrier system to transport peptides across the intestinal barrier and therefore present a useful tool for the development of novel drugs.
Furthermore, to mimic the complex structure and physiology of the gut including the presence of multiple different cell types, the Caco-2 cell line was replaced by primary intestinal cells to set up a de novo tissue model. To that end, intestinal crypts including undifferentiated stem cells and progenitor cells were isolated from human small intestinal tissue samples (jejunum) and expanded in vitro in organoid cultures. Cells were cultured on the decellularized porcine gut matrix in co-culture with intestinal myofibroblasts. These novel tissue models were maintained under either static or dynamic conditions.
Primary intestinal epithelial cells formed a confluent monolayer including the major differentiated cell types positive for mucin (goblet cells), villin (enterocytes), chromogranin A (enteroendocrine cells) and lysozyme (paneth cells). Electron microscopy images depicted essential functional units of an intact epithelium, such as microvilli and tight junctions. FITC-dextran permeability and TEER measurements were used to assess tightness of the cell layer. Models showed characteristic transport activity for several reference substances. Mechanical stimulation of the cells by a dynamic culture system had a great impact on barrier integrity and transporter activity resulting in a tighter barrier and a higher efflux transporter activity.
In Summary, the use of primary human intestinal cells combined with a biological decellularized scaffold offers a new and promising way to setup more physiological intestinal in vitro models. Maintenance of primary intestinal stem cells with their proliferation and differentiation potential together with adjusted culture protocols might help further improve the models. In particular, dynamic culture systems and co culture models proofed to be a first crucial steps towards a more physiological model. Such tissue models might be useful to improve the predictive power of in vitro models and in vitro in vivo correlation (IVIVC) studies. Moreover, these tissue models will be useful tools in preclinical studies to test pharmaceutical substances, probiotic active organisms, human pathogenic germs and could even be used to build up patient-specific tissue model for personalized medicine.
The enteric nervous system (ENS) innervates the gastrointestinal (GI) tract and controls central aspects of GI physiology including contractility of the intestinal musculature, glandular secretion and intestinal blood flow. The ENS is composed of neurons that conduct electrical signals and of enteric glial cells (EGCs). EGCs resemble central nervous system (CNS) astrocytes in their morphology and in the expression of shared markers such as the intermediate filament protein glial fibrillary acidic protein (GFAP). They are strategically located at the interface of ENS neurons and their effector cells to modulate intestinal motility, epithelial barrier stability and inflammatory processes. The specific contributions of EGCs to the maintenance of intestinal homeostasis are subject of current research.
From a clinical point of view EGC involvement in pathophysiological processes such as intestinal inflammation is highly relevant. Like CNS astrocytes ECGs can acquire a reactive, tissue-protective phenotype in response to intestinal injury. In patients with chronic inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis, alterations in the EGC network are well known, particularly a differential expression of GFAP, which is a hallmark of reactive gliosis in the CNS.
With increasing recognition of the role of EGCs in intestinal health and disease comes the need to study the glial population in its complexity. The overall aim of this thesis was to comprehensively study EGCs with focus on the reactive GFAP-expressing subpopulation under inflammatory conditions in vivo and in vitro. In a first step, a novel in vivo rat model of acute systemic inflammation mimicking sepsis was employed to investigate rapidly occuring responses of EGCs to inflammation. This study revealed that within a short time frame of a few hours, EGCs responded to the inflammation with an upregulation of Gfap gene expression. This inflammation-induced upregulation was confined to the myenteric plexus and varied in intensity along the intestinal rostro-caudal axis. This highly responsive myenteric GFAP-expressing EGC population was further characterized in vivo andin vitro using a transgenic mouse model (hGFAP-eGFP mice). Primary purified murine GFAP-EGC cultures in vitro were established and it was assessed how the transcriptomic and proteomic profiles of these cells change upon inflammatory stimulation. Here, myenteric GFAP-EGCs were found to undergo a shift in gene expression profile that predominantly affects expression of genes associated with inflammatory responses. Further, a secretion of inflammatory mediators was validated on protein level. The GFAP+ subpopulation is hence an active participant in inflammatory pathophysiology. In an acute murine IBD model in vivo, GFAP-EGCs were found to express components of the major histocompatibility complex (MHC) class II in inflamed tissue, which also indicates a crosstalk of EGCs with the innate and the adaptive lamina propria immune system in acute inflammation.
Taken together, this work advances our knowledge on EGC (patho-)physiology by identifying and characterizing an EGC subpopulation rapidly responsive to inflammation. This study further provides the transcriptomic profile of this population in vivo and in vitro, which can be used to identify targets for therapeutic intervention. Due to the modulating influence of EGCs on the intestinal microenvironment, the study further underlines the importance of integrating EGCs into in vitro test systems that aim to model intestinal tissues in vitro and presents an outlook on a potential strategy.
Kinetic assessment by in vitro approaches - A contribution to reduce animals in toxicity testing
(2015)
The adoption of directives and regulations by the EU requires the development of alternative testing strategies as opposed to animal testing for risk assessment of xenobiotics. Additionally, high attrition rates of drugs late in the discovery phase demand improvement of current test batteries applied in the preclinical phase within the pharmaceutical area. These issues were taken up by the EU founded 7th Framework Program “Predict-IV”; with the overall goal to improve the predictability of safety of an investigational product, after repeated exposure, by integration of “omics” technologies applied on well established in vitro approaches. Three major target organs for drug-induced toxicity were in focus: liver, kidney and central nervous system. To relate obtained dynamic data with the in vivo situation, kinetics of the test compounds have to be evaluated and extrapolated by physiologically based pharmacokinetic modeling.
This thesis assessed in vitro kinetics of the selected test compounds (cyclosporine A, adefovir dipivoxil and cisplatinum) regarding their reliability and relevance to respective in vivo pharmacokinetics. Cells were exposed daily or every other day to the test compounds at two concentration levels (toxic and non-toxic) for up to 14 days. Concentrations of the test compounds or their major biotransformation products were determined by LC-MS/MS or ICP-MS in vehicle, media, cells and plastic adsorption samples generated at five different time-points on the first and the last treatment day.
Cyclosporine A bioaccumulation was evident in primary rat hepatocytes (PRH) at the high concentration, while efficient biotransformation mediated by CYP3A4 and CYP3A5 was determined in primary human hepatocytes (PHH) and HepaRG cells. The lower biotransformation in PRH is in accordance with observation made in vivo with the rat being a poor model for CYP3A biotransformation. Further, inter-assay variability was noticed in PHH caused by biological variability in CYP3A4 and CYP3A5 activity in human donors. The inter-assay variability observed for PRH and HepaRG cells was a result of differences between vehicles regarding their cyclosporine A content. Cyclosporine A biotransformation was more prominent in HepaRG cells due to stable and high CYP3A4 and CYP3A5 activity. In addition, in vitro clearances were calculated and scaled to in vivo. All scaled in vitro clearances were overestimated (PRH: 10-fold, PHH: 2-fold, HepaRG cells: 2-fold). These results should be proven by physiologically-based pharmacokinetic modeling and additional experiments, in order to verify that these overestimations are constant for each system and subsequently can be diminished by implementation of further scaling factors.
Brain cell cultures, primary neuronal culture of mouse cortex cells and primary aggregating rat brain cells, revealed fast achieved steady state levels of cyclosporine A. This indicates a chemical distribution of cyclosporine A between the aqueous and organic phases and only minor involvement of biological processes such as active transport and biotransformation. Hence, cyclosporine A uptake into cells is presumably transport mediated, supported by findings of transporter experiments performed on a parallel artificial membrane and Caco-2 cells. Plastic adsorption of cyclosporine A was significant, but different for each model, and should be considered by physiologically based pharmacokinetic modeling.
Kinetics of adefovir dipivoxil highlights the limits of in vitro approaches. Active transporters are required for adefovir uptake, but were not functional in RPTECT/TERT1. Therefore, adefovir uptake was limited to passive diffusion of adefovir dipivoxil, which itself degrades time-dependently under culture conditions.
Cisplatinum kinetics, studied in RPTEC/TERT1 cells, indicated intracellular enrichment of platinum, while significant bioaccumulation was not noted. This could be due to cisplatinum not reaching steady state levels within 14 days repeated exposure. As shown in vivo, active transport occurred from the basolateral to apical side, but with lower velocity. Hence, obtained data need to be modeled to estimate cellular processes, which can be scaled and compared to in vivo.
Repeated daily exposure to two different drug concentrations makes it possible to account for bioaccumulation at toxic concentrations or biotransformation/extrusion at non-toxic concentrations. Potential errors leading to misinterpretation of data were reduced by analyses of the vehicles as the applied drug concentrations do not necessarily correspond to the nominal concentrations. Finally, analyses of separate compartments (medium, cells, plastic) give insights into a compound’s distribution, reduce misprediction of cellular processes, e.g. biotransformation, and help to interpret kinetic data. On the other hand, the limits of in vitro approaches have also been pointed out. For correct extrapolation to in vivo, it is essential that the studied in vitro system exhibits the functionality of proteins, which play a key role in the specific drug induced toxicity. Considering the benefits and limitations, it is worth to validate this long-term treatment experimental set-up and expand it on co-culture systems and on organs-on-chips with regard to alternative toxicity testing strategies for repeated dose toxicity studies.
Die Epithelzellen des renalen proximalen Tubulus resorbieren große Mengen an Wasser, Glucose und weiteren wertvollen Substanzen aus dem Primärharn, um deren Ausscheidung zu verhindern. Weiterhin sekretieren sie harnpflichtige Substanzen in den Primärharn und sind in der Lage, in die Zelle aufgenommene Substanzen enzymatisch umzusetzen. Diese Funktionen machen den renalen proximalen Tubulus zu einer wichtigen Einheit für die Nie-renfunktion. Sie führen aber auch zu einer hohen Empfindlichkeit gegenüber toxischen Effek-ten von Fremdstoffen. Daher ist ein In-vitro-Modell des renalen proximalen Tubulusepithels sowohl für die Erforschung physiologischer und pathologischer Mechanismen als auch zur Testung der Toxizität von Substanzen, insbesondere neuen Arzneimitteln, bedeutend. Ein weiteres Forschungsfeld, für das ein In-vitro-Gewebe von großem Nutzen wäre, ist die Ent-wicklung von bioartifiziellen Nierenersatzsystemen. Aufgrund Spezies-spezifischer Unterschiede, z.B. in der Expression von Transportproteinen und Enzymen, ist ein Modell mit humanen Zellen anzustreben. Bisher besteht jedoch ein Mangel an Modellen, die das renale proximale Tubulusepithel für die oben genannten An-wendungsbereiche adäquat abbilden. Das Ziel dieser Arbeit war deshalb der Aufbau eines humanen In-vitro-Modells des renalen proximalen Tubulus unter Verwendung von humanen Nierenzellen (human kidney-derived cells, hKDCs), die Eigenschaften renaler Vorläuferzellen aufweisen. In Kombination mit die-sen Zellen wurden verschiedene Kultursubstrate getestet. Dabei zeigte sich, dass die Zellen sowohl in Zellkulturplatten als auch auf Kollagen-Typ-I-beschichteten Insertmembranen mehrschichtig wachsen, ohne die typische Morphologie renaler proximaler Tubuluszellen auszubilden. In einem dreidimensionalen Kollagen-Typ-I-Hydrogel bildeten die hKDCs hin-gegen tubuläre bzw. zystäre Strukturen mit einer kubischen bis hochprismatischen Morpho-logie. Da für die oben erwähnten Anwendungsbereiche jedoch eine planare Zellschicht benö-tigt wird, erfolgte die Testung weiterer biologischer Matrices. Diese waren die Small intestinal submucosa (SIS) und das Biological vascularized scaffold (BioVaSc). Beide ließen sich aus porcinem Dünndarm herstellen, wobei bei der SIS die Mucosa sowie das Mesenterium ent-fernt wurden. Bei der BioVaSc handelt es sich um ein Darmsegment mit erhaltenem Ge-fäßsystem, dass zur Perfusion genutzt wird. Nach ihrer Kultur auf der SIS wiesen die hKDCs das typische Wachstum und die charakteris-tische Morphologie des renalen proximalen Tubulusepithels auf. Dazu gehören die Kontakt-hemmung, die das einschichtige Wachstum ermöglicht, die kubisch bis hochprismatische Morphologie sowie die Bildung eines Bürstensaums an der apikalen Zellmembran. Anhand einer Kollagen-Typ-IV- und einer Alcianblau-Färbung ließ sich die Bildung einer Basalmemb-ran an der Grenze zur SIS nachweisen. Bürstensaum- und Basalmembranbildung zeigten die zelluläre Polarisierung. Weiterhin waren typische Markerproteine renaler proximaler Tu-buluszellen wie N-Cadherin und Aquaporin-1 immunhistochemisch, zum Teil deutlich stärker als bei den Ausgangszellen, nachweisbar. Dies belegt einen positiven Einfluss der extrazellu-lären Matrixkomponenten der SIS auf die Ausbildung von Charakteristika des renalen proxi-malen Tubulusepithels. Die Albuminaufnahme als spezifische Funktion war ebenfalls nach-weisbar. Die molekularen Veränderungen der hKDCs während der Kultivierung auf der SIS ließen sich weiterhin mittels Raman-Spektroskopie bestätigen. Aufgrund der starken Interak-tion zwischen Tubulusepithel und umgebenden Kapillarnetzwerk wurde weiterhin die Co-Kultur mit Endothelzellen etabliert. Für den Vergleich der hKDCs mit einer etablierten humanen Zelllinie renaler proximaler Tu-buluszellen wurde die HK-2-Zelllinie verwendet. Mit dieser Zelllinie ließen sich die Ergebnisse der hKDCs jedoch nicht reproduzieren, was auf die fehlende Sensitivität der transformierten Zelllinie auf die Substrateigenschaften zurückzuführen ist. In der dynamischen Kultur mit der BioVaSc als Matrix waren ein inhomogenes Wachstum sowie eine variierende Markerexpression zu beobachten. Die ließ sich vor allem auf den starken Einfluss der Aussaatdichte sowie die Festigkeit der Matrix zurückführen. Bei einer erfolgreichen Optimierung der Kultur kann dieses Modell jedoch für komplexere Studien in der pharmakologischen Entwicklung nützlich sein. Mit der Kombination aus hKDCs und SIS ist es gelungen, eine einzelne, durchgängige Zell-schicht zu generieren, die wichtige Charakteristika des renalen proximalen Tubulusepithels aufweist. Weitere Untersuchungen sind nun nötig, um die Funktionalität des Modells weiter-gehend zu charakterisieren (z.B. der Transport von Substanzen und Sensitivität gegenüber toxischen Substanzen). Anschließend kann es für die spezifischen Anwendungen weiterentwickelt werden.