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- Biopharmazie (17)
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Oral administration of drugs is the most common, convenient, safest and economical route of drug administration. There is lack of established tools to study the function of transporters in the intestinal absorption of drugs. Because of its favorable physico-chemical, pharmacokinetic and pharmacodynamic characteristics, trospium could be potentially used as a probe substrate to study the function of drug transporters. Therefore, this study was conducted to examine the suitability of trospium chloride as a probe drug to study the function of multidrug transporters in the human body. To this end, two randomized, controlled, four-period, cross-over pharmacokinetic drug interaction studies of oral and intravenous trospium with co-medication of oral clarithromycin or ranitidine were performed in 24 healthy subjects to mechanistically characterize the role of P-gp, OATP1A2, OCT1, OCT2, MATE1 and MATE2-K in the absorption and disposition of trospium. The contribution of the drug transporters in the absorption and disposition of trospium were examined in isolated systems using in vitro uptake and inhibition assays in transporter transfected human cell lines.
OCT1 (Vmax = 0.8 ± 0.1 nmol/min × mg) is a high capacity transporter of trospium compared to OCT2 (Vmax = 0.04 ± 0.01 nmol/min × mg). But the OCT2 (Km = 0.5 ± 0.1 µM) transporter demonstrated a high affinity in the transport of trospium compared to OCT1 (Km = 17.4 ± 2.1 µM). OCT1 genetic alleles *2, *3, *4 and *7 resulted in significant loss of activity and the alleles *5 and *6 caused complete loss of uptake of trospium. The common OCT2 genetic allele Ser270 caused slight but significant increase in activity of OCT2.
Ranitidine inhibits OCT1 (IC50 = 186 ± 25 µM), MATE1 (IC50 = 134 ± 37 µM) and MATE2-K (IC50 = 35 ± 11 µM)-mediated uptake of trospium in vitro. But it is a weak inhibitor of OCT2 transporter (IC50 = 482 ± 105 µM). Using FDA and EMA in vitro to in vivo extrapolation models, ranitidine was predicted to have a potential inhibition effect on intestinal OCT1 ([I]2/IC50 ~40), renal MATE1 ([I]1/IC50 ~0.02) and MATE2-K ([I]1/IC50 ~0.1) transporters in vivo. Clarithromycin was predicted to cause DDI by inhibiting P-gp-mediated efflux of trospium at the intestine ([I]2/IC50 of ~310) and hepatocytes ([I]3/IC50 ~1). Therefore, co-medication of oral clarithromycin was expected to result in an increase in oral absorption and hepatic clearance of trospium but not changes in distribution volume.
In healthy subjects, oral trospium is slowly (MAT ~10 h) and poorly (F ~10 %) absorbed from the jejunum and cecum/ascending colon, widely distributed into the body (Vss = 5 - 6 l/kg) and slowly eliminated (t1/2 = 9 - 10 h) majorly via renal glomerular filtration and tubular secretion (CLR ~500 ml/min). After co-medication of clarithromycin (inhibitor of P-gp), on the contrary to our IVIVE prediction, we found a non-expected but significant expansion of the shallow and deep distribution spaces for trospium by ~27 %. A single dose administration of trospium with co-medication of ranitidine (inhibitor of OCT1) resulted in no effect on the intestinal absorption of trospium. But the renal clearance of trospium decreased slightly (15 %) but significantly.
Intravenously administered trospium (2 mg TC) might be a suitable probe drug to evaluate the effects of a P-gp inhibitor on distribution of a drug. Oral trospium chloride can be selected for DDI studies with new chemical entities (NCE) with predicted inhibitory potential on OCT1 and P-gp and which are available after oral absorption along the small intestine and in the cecum/ascending colon. Another kind of application of trospium chloride might be pharmacogenomics studies in subjects with functionally relevant polymorphisms of P-gp and OCT1 or in patients with suspected transport failure due to intestinal diseases. The function of the efflux transporters MATE1 and MATE2-K in the PTC of the kidneys can be well assessed with the probe drug trospium by measuring its renal clearance.
The aim of the present dissertation was to investigate the biological and chemical potential of two European mushroom species: Fomitopsis betulina and Calvatia gigantea. For this purpose, different extracts of both fungi were tested for: antimicrobial, antifungal, cytotoxic, in vitro wound healing, and anti-adhesive properties. Bioassay-guided fractionation led to the isolation of bioactive compounds, altogether 20 compounds were isolated and identified. The compounds were obtained from the ethyl acetate extracts, they included triterpenes, sterols and aromatic compounds. The separated substances from both fungi were proved for biological activities, some of them showed antimicrobial and cytotoxic activities.
Infections with Helicobacter pylori are a global challenge that affects both developed and developing countries. This infection is currently treated using multiple antimicrobials that are mostly absorbed after oral administration and subsequently secreted into the gastric lumen. The eradication rates from the different therapeutic regimens, however, are declining nowadays, primarily due to high antibiotic resistance and possibly the mode of drug delivery. H. pylori is commonly found adhering to epithelial cells, and therefore, intragastric drug delivery may be a more direct treatment option. In this work, we developed a new strategy for the local eradication of H. pylori within the stomach.
Initial in vitro experiments revealed that penicillin G shows promising antibiotic activity against resistant strains of H. pylori with MIC values of 0.125 µg/mL. To provide luminal concentrations above the MIC for an extended time, we decided to follow two different formulation strategies: effervescent granules and HPMC-based hydrogel matrix tablets. Among the granule formulations, only one batch was stable and demonstrated excellent performance with respect to drug content, effervescent action, and drug release. It was therefore selected for further in vitro studies. All matrix tablets showed the desired tablet quality requirements and drug release was scalable in vitro by the HPMC concentration.
In order to quantify PGS in various formulations and media, an HPLC method was developed and validated. Due to the stability concerns, the degradation behavior of PGS was studied at different pH. PGS was found to be unstable at acidic pH values, but its stability was higher at more neutral pH values. Sufficient stability was exhibited at pH values above pH 4.5. Due to the instability of PGS in acidic media, alkalizers were added to the matrix tablets to prevent the degradation of the drug within the tablet. Among the alkalizers tested, NaHCO3 showed the most promising results as it significantly enhanced the stability within the matrix and also the concentration of PGS in the dissolution media. The stabilizing effect was caused mainly by the modulation of the microenvironmental pH rather than a pH change in the dissolution media. As a result, these matrix tablets were selected for further in vitro characterization.
In order to guide formulation development, a flow-through model (FTM), which was able to simulate various physiological conditions of the gastric environment, was developed and applied. In contrast to compendial dissolution methods, the FTM allowed studying the effect of gastric secretion, mixing and emptying on the gastric concentration of the drug in vitro. It could be shown that the granules generated a high initial concentration, which decreased over time. On the contrary, the matrix tablets did not provide such a profile due to the absence of pressure events in the model. Further investigations of the matrix tablets in a dissolution stress test device revealed faster drug release if pressure events of physiological relevance are simulated.
In the last part of this thesis, the two formulation concepts were compared in vivo by using the salivary tracer technique. For this purpose, caffeine was used as a model drug. The in vivo investigations suggested that granules administered in a fed state demonstrated longer gastric retention than in a fasted state. In a fed state, effervescent granules provided longer gastric retention of caffeine in comparison to the matrix tablets. Interestingly, the administration of the granules together with 240 mL of tap water provided an even better gastric retention of caffeine than the smaller volume (20 mL). Additional MRI investigations after 4 h of tablets’ intake revealed that the matrix tablets were already disintegrated in vivo.
In conclusion, effervescent granules dosed after food are expected to better maintain intragastric drug concentration over an extended period compared to matrix tablets. Moreover, the carbon dioxide generated after disintegration supports the mixing of the drug with the chyme and thus, provides a uniform distribution of the drug. By this, bacterial sanctuary sites within the stomach can be avoided. The major challenge could be the stability of PGS in acidic media. This problem could be addressed via concomitant administration of PPIs. H2 blockers could also be recommended to address nocturnal acid-breakthrough during the mid-night. In combination with an acid-reducing agent, PGS granule formulations alone or part of the treatment regimens could enable the local eradication of H. pylori directly within the stomach.
Analysis and Reduction of Cellular Heterogeneity in Strain Optimization of Bacillus licheniformis
(2021)
Bacillus species invest substantial resources in inherent cellular processes for pre-adaptation to environmental changes, many of which are dispensable in the controlled environment of industrial bioprocesses. The underlying physiological mechanisms are well characterized in B. subtilis, but only little is known about these processes in the closely related B. licheniformis. Moreover, experimental conditions in previous studies differ from industrial settings in most parameters, foremost in batch cultures or plate-based analysis over fed-batch processes. In this thesis, cellular heterogeneity was analyzed in B. licheniformis in optimized, nutrient-rich media in batch and fed-batch cultivations. Systematic inactivation of genes involved in biofilm formation and synthesis of the flagellar apparatus or global regulators thereof resulted in higher protein production and provided new insights into biofilm formation and cellular heterogeneity in this strain.
Die Therapie von Erkrankungen des hinteren Auges erfolgt heute hauptsächlich durch die intravitreale Injektion von Lösungen, Suspensionen oder Implantaten. Um neue intravitreale Arzneiformen zu entwickeln werden in der präklinischen Phase neben In vitro-Untersuchungen zur Wirkstofffreisetzung auch In vivo-Studien an Tieren verwendet. Die Physiologie des Auges der verwendeten Tiere weicht jedoch von denen des humanen Glaskörpers ab, weshalb die Übertragbarkeit der Ergebnisse teilweise kontrovers diskutiert wird. Durch eine Kombination dieser in vivo-Studien mit biorelevanteren In vitro-Freisetzungsmodellen könnte ein besseres Verständnis für das Verhalten von intravitrealen Arzneiformen erhalten werden.
In dieser Arbeit wurde die EyeFlowCell entwickelt, bei der zentral der humane Glaskörper durch ein künstliches Gel simuliert wird. In dieses Glaskörpersubstitut injizierte Arzneiformen können hinsichtlich ihrer Wirkstofffreisetzung unter verschiedenen Aspekten charakterisiert werden…
Synthesis and evaluation of pseudosaccharin amine derivatives as potential elastase inhibitions
(2006)
Elastase is a serine protease which by definition is able to solubilize elastin by hydrolytic cleavage.Human Leukocyte Elastase, HLE (EC 3.4.21.37), is involved in deseases such as adult respiatory distress syndrome, pulmonary emphysema, smoking related chronic bronchitits, ischemic-reperfusion injury and rheumatoid arthritis. Hence, the elastase inhibitors have clinical utility in these diseases. Heterocyclic compounds are one of the most important classes of the elastase inhibitiors. In the present work different pseudosaccharin amine derivatives were synthesized and tested against the elastase. The synthesis of pseudosaccharin amine dervatives was carried out from the amines and(1,1-dioxobenzo[d]isothiazol-3-ylsulfanyl)acetonitrile in different solvents. Futhermore, the pseudosaccharin amines were obtained by refluxing the thiosaccarinates in absolute acetic acid. The reaction of 3-ethoxybenzo[d]isothiazole 1,1-dioxide with different amines in dioxane under reflux resulted into the desired pseudosaccharin amine derivatives in higher yields. Pseudosaccharin chloride was also used in the synthesis of these derivatives.A detail study of the synthesis of pseudosaccharin amine dervatives from the above differnt routes is described. Peptides were also synthesized by using the mixed anhydride method. The ester, acid, amide and peptide derivatives were tested against the Porcine Pancreatic Elastase (PPE) and Human Leukocyte Elastase (HLE). The esters were found to be the reversible inhibitors of HLE. The process of the PPE inhibion by cyanomethyl(2S)-2-(1,1-dioxobenzo[d]isothiazol-3-ylamino)-3-methylbutanoate was studied. Michaelis-Menten curve and Lineweaver-Burk double reciprocal plot were constructed in order to study the kinetic of this reaction. The compounds showing high inhibition of HLE were further stuied for determination of their inhibitory constant(Ki). The esters were found to be the higly active compounds against HLE. The cyanomethyl(2S)-2-(1,1-dioxobenzo[d]isothiazol-3-ylamino)-3-methylbutanoate and cyanomethyl(2S,3S)-2-(1,1-dioxobenzo[d]isothiazol-3-ylamino)-3-methylpentanoate showed the competitive reversible inhibition of HLE.The cyanomethyl(2S,3S)-2-(1,1-dioxobenzo[d]isothiazol-3-ylamino)-3-methylpentanoate is highly potent inhibitor of HLE. The possible mechanism of inhibition of elastase by these compounds is discussed. Molecular modelling of some of the ester derivatives is also discussed.
Pharmaceutical residues are found in increasing concentrations in the environment and in potable water where they have verifiable effects on aquatic life. Conventional methods for water treatment are not able to sufficiently abate these generally stable compounds. It was found that physical plasma generated directly in water can degrade several of these recalcitrant organic pollutants. Studies on the basic plasma chemical processes for the model system of phenol showed that the degradation is primarily caused by hydroxyl radicals. This was confirmed by reaction chemistry and spin trap enhanced electron paramagnetic resonance spectroscopy (EPR). The degradation of diclofenac and its by-products were investigated in detail to perform a first risk-assessment of the new technology. Findings are not limited to the application of plasma but applicable to other advanced oxidation processes (AOP) that are based on the generation of hydroxyl radicals as well. Additionally, pulsed corona plasma and pulsed electric fields were assessed for their capacity to kill Legionella pneumophila in water. Whereas it was possible to kill L. Pneumophila with both methods, plasma treatment resulted in an enhanced bacterial killing. Therefore, advanced oxidation processes (AOP) and plasma treatment in particular are some of the few feasible approaches to decompose recalcitrant compounds in water.
There is a growing interest in the application of non-thermal atmospheric pressure plasma for the treatment of wounds. Due to the generation of various ROS and RNS, UV radiation and electric fields plasma is a very promising tool which can stimulate skin and immune cells. However, not much is known about the mammalian cell responses after plasma treatments on a molecular level. The present work focusses on the impact of plasma on cell signaling in the human keratinocyte cell line HaCaT by using the methods DNA microarray, qPCR, ELISA and flow cytometry. Here, cell signaling mediators such as cytokines and growth factors which could promote wound healing by enhancing angiogenesis, reepithelization, migration and proliferation were of major interest. Additionally, the crosstalk between keratinocytes and monocytes was studied using a co-culture. For the first time extensive investigations on the impact of plasma on cell signaling in human keratinocytes were conducted. The most prominent cytokines and growth factors which were regulated by plasma at gene and protein level were VEGF-A, GM-CSF, HB-EGF, IL-8, and IL-6. The latter was not activated due to the JAK/STAT-pathway but probably by a combined activation of MAPK- and PI3K/Akt-pathways. By the use of conditioned medium it was found out that ROS and RNS generated directly after plasma treatment induced larger effects on cell signaling in keratinocytes than the subsequently secreted growth factors and cytokines. Furthermore, monocytes and keratinocytes hardly altered their secretion profiles in co-culture. From these results it is deduced that the plasma generated reactive species are the main actors during cell signaling. In order to differentiate the impact of ROS and RNS on the cellular response the ambience of the plasma effluent was controlled, varying the ambient gas composition from pure nitrogen to pure oxygen. Thereby a first step towards the attribution of the cellular response to specific plasma generated reactive species was achieved. While IL-6 expression correlated with ROS generated by the plasma source, the cell signaling mediators VEGF-A, GM-CSF and HB-EGF were significantly changed by RONS. Above all hydrogen peroxide was found to play a dominant role for observed cell responses. In summary, plasma activates wound healing related cell signaling mediators as cytokines and growth factors in keratinocytes. It was also shown that the generated reactive species mainly induced cell signaling. For the first time cell responses can be correlated to ROS and RONS in plasma treated cells. These results underline the potential of non-thermal atmospheric pressure plasma sources for their applications in wound treatment.
Lungenkrebs ist mit rund 40.000 Todesfällen pro Jahr die häufigste Krebstodesursache in Deutschland. Daher ist es nötig neue Behandlungsmethoden zu entwickeln, die gezielt am Tumor angreifen und möglichst geringe Nebenwirkungsraten aufweisen. Dazu eignet sich möglicherweise das inhalative magnetische Drug Targeting, bei dem superparamagnetische Aerosoltröpfchen als Drug Carrier dienen, welche nach der Inhalation mittels eines extern angelegten Gradientenfeldes gezielt am Wirkungsort angereichert werden. Im Rahmen dieser Arbeit wurde untersucht, ob es möglich ist superparamagnetische Aerosole mit Hilfe von Magnetfeldgradienten von ihrer Flugbahn abzulenken, um sie an einer definierten Abscheidefläche vor dem Magneten abzuscheiden. Dabei wurden einige Parameter, wie die Kerngröße der magnetischen Nanopartikel, der Tröpfchendurchmesser, die Gradientenfeldstärke, die Ferrofluidkonzentration und die Strömungsgeschwindigkeit der Luft variiert, um deren Einfluss auf die Abscheidung zu beobachten. Die Grundlage dieses magnetischen Aerosols bildet ein Ferrofluid, welches mittels Ammoniak aus Eisen(II)- und Eisen(III)-chlorid-Lösung gefällte Magnetit-Nanopartikel enthält, die durch eine Citrathülle vor Agglomeration und Sedimentation geschützt werden. Nach dem Vernebeln mit zwei Geräten, die auf unterschiedlichen Verneblungsprinzipien beruhen, sind die magnetischen Nanopartikel im Aerosoltröpfchen eingeschlossen. Die mittels Laserdiffraktometrie bestimmten mittleren Massendurchmesser betragen dabei zwischen 2,5 µm mit dem Pari Boy und 5 µm mit dem eFlow. Diese sollten durch Zusatz von Substanzen, die die Oberflächenspannung senken (Cremophor RH 40 und Ethanol), die Viskosität erhöhen (Glycerol, Cremophor RH 40 und Ethanol) oder den Dampfdruck erhöhen (Ethanol) gesenkt werden. Deutliche Effekte zeigen sich insbesondere bei Zugabe von 20 % Cremophor RH 40 und 40 % Ethanol zum Ferrofluid. Dadurch konnten die Durchmesser auf jeweils 2 µm beim Pari Boy und circa 3,4 und 3,9 µm beim eFlow reduziert werden. Die prozentuale Abscheidung des Ferrofluids wurde in verschiedenen Magnetfeldgeometrien unterschiedlicher Gradientenfeldstärke untersucht. Die Variation der Tröpfchengröße des superparamagnetischen Aerosols führt zu unterschiedlichen Ausmaßen der Ferrofluidabscheidung. Tatsächlich ist die Abscheidung des Ferrofluides nach dem Vernebeln mit dem eFlow, dessen Tröpfchendurchmesser etwa doppelt so groß wie der des Pari Boy ist, wesentlich höher. Während mit dem Pari Boy vom 1 molaren Ferrofluid im Gradientenfeld zweier gleichpoliger Stabmagnete (r x l = 15 x 25 mm) mit einer Remanenz von 1450 mT nur circa 30 % des Aerosols impaktiert werden kann, sind es mit dem eFlow unter gleichen Versuchsbedingungen etwa 80 %. Das liegt einerseits an der größeren Magnetitmasse im Tröpfchen und andererseits an der geringeren Aerosolgeschwindigkeit. Je stärker das verwendete Gradientenfeld war, desto mehr Ferrofluid konnte impaktiert werden. Desweiteren wurde die Abscheidung verschieden konzentrierter Ferrofluide (0,5; 1, 2 und 6 M) untersucht. Diese war umso größer, je konzentrierter das Ferrofluid war. Unterschiedliche Strömungsgeschwindigkeiten konnten durch Verwendung einer Vakuumpumpe untersucht werden. Es wurden zwei sehr kleine Strömungsgeschwindigkeiten (0,03 und 0,1 m/s) eingestellt, um möglichst nahe an die realen Strömungsgeschwindigkeiten in den terminalen Bronchien zu kommen. Dabei zeigte sich, dass die prozentuale Ferrofluidabscheidung bei langsamen Strömungsgeschwindigkeiten besonders hoch ist. Allerdings weisen die in dieser Arbeit verwendeten Magnete in größerer Entfernung nur noch sehr schwache Gradienten auf und sind daher für eine Anwendung an der menschlichen Lunge nicht geeignet. Die Einführung eines Modellimplantats in die Versuchsröhre sollte die Abscheidung des Ferrofluides erhöhen. Die erzielte Erhöhung der Ferrofluidabscheidung ist allerdings unbefriedigend. Die Simulation mit Mathematica® hat ergeben, dass die experimentelle Ferrofluidabscheidung geringer ist als die theoretische Abscheidung. Beim Pari Boy und 0,1 m/s unterscheiden sie sich um maximal 10 %. Beim eFlow beträgt die Differenz bis zu 8 %. Die Abweichungen zur Simulation werden durch Sedimentation des Ferrofluides verursacht. Diese ist stärker ausgeprägt als in der Berechnung, da sich die Luft beim Vernebeln durch Verdunsten von Wasser und Ausdehnung der Luft aufgrund des Joule-Thomson-Effektes abkühlt. Die kalte Luft bewegt sich nach unten und reißt das Ferrofluid mit herab. Es konnte gezeigt werden, dass sich superparamagnetische Aerosole mit Hilfe von magnetischen Gradientenfeldern gezielt in definierten Bereichen abscheiden lassen. Das Ausmaß der Abscheidung lässt sich durch Erhöhung des Gradienten und des magnetischen Momentes steigern. Mit größeren Permanentmagneten oder starken Elektromagneten ist auch ein pulmonales Drug Targeting an der humanen Lunge denkbar.
In drei verschiedenen Teilanalysen wurden Erkenntnisse über die Wirksamkeit von Interventionsmaßnahmen gegen das Vorkommen und die Verbreitung von ESBL/AmpC-bildenden E. coli in der Hühnermast gewonnen. Literaturdaten und praktische Laborergebnisse, die teilweise selbst erhoben wurden, flossen in ein mathematisches Modell ein, um die Auswirkungen von Haltungsparametern und konkreten Interventionsmaßnahmen prädiktiv berechnen zu können. Die zusammengefassten Ergebnisse zeigen einen Einfluss der Maßnahmen „Competitive Exclusion“, „Reinigung und Desinfektion“ sowie den Haltungsparametern „Rasse“, „geringe Besatzdichte“ und „erhöhte Einstreumenge“ auf das Vorkommen von bestimmten ESBL/AmpC-bildenden E. coli. Zusätzlich zu den Einzelmaßnahmen wurden im Modell mehrere Kombinationen getestet, wobei zwei unterschiedlichen Szenarien verwendet wurden, entweder der Stall oder die Eintagsküken waren zu Beginn der Mastperiode positiv. Diese Kombinationen ergaben eine deutliche Reduktion der resistenten E. coli in den infizierten Tieren, in deren Ausscheidungen und in der Einstreu. In diesem Zusammenhang wären Daten aus tierexperimentellen Studien zu kombinierten Maßnahmen interessant. Weitere wissenschaftliche Ergebnisse könnten zu einem optimierten Modell beitragen, damit es die realen Bedingungen besser widerspiegelt. Zu einer weiteren Präzisierung könnte zum Beispiel die dezidierte mathematische Berechnung des Wachstums von resistenten E. coli in den unterschiedlichen Teilen des Gastrointestinaltrakts des Huhns und in der Einstreu führen, unter Berücksichtigung von pH-Wert und Temperatur. Ungeachtet dessen bietet die vorliegende Version des Modells eine nützliche Unterstützung bei der Vorhersage der Auswirkung unterschiedlicher Maßnahmen auf das Auftreten und die Verbreitung von ESBL/AmpC-bildenden E. coli in Masthuhnbetrieben. Diese Ergebnisse können zu einer umfassenden Quantitativen mikrobiologischen Risikobewertung (QMRA) beitragen, mittels derer die Effizienz von Risikominderungsmaßnahmen in der gesamten Broilerproduktionskette, von der Brüterei und Aufzucht über die Mast, Schlachtung, Verarbeitung und den Einzelhandel bis hin zum Verzehr - from farm to fork, bestimmt werden kann.