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In vitro assays play a crucial role in the biopharmaceutical assessment of drugs. During the past two decades, biorelevant media became an indispensable tool to forecast the in vivo solubility and dissolution of pharmaceutical drug candidates, and to assess absorption risks like low solubility or drug precipitation. Nevertheless, in vitro set-ups are still a simplification of the conditions in the human GI tract. This thesis aimed to shed light on some of the remaining open questions, aiming at providing a better understanding of the effects of biorelevant media on solubility, dissolution, and precipitation processes, and providing guidance for a more streamlined usage in the future. The results of this work can be outlined in brief as follows: First, a new design of experiment-based method development was introduced which increased the robustness and accuracy of derivative UV spectrophotometric methods for drug quantification in biorelevant precipitation assays. Second, based on this new approach, the impact of SIF powder aging on the supersaturation and precipitation behavior of the model drug ketoconazole was investigated. Recommendations on the use of biorelevant media for precipitation assays were developed to further improve the reproducibility of transfer experiments and to enhance data reliability. Third, it was investigated under which circumstances the physiological bicarbonate buffer should be applied to Fasted State Simulated Intestinal Fluid medium for in vitro solubility, dissolution, and precipitation testing to resemble the in vivo conditions.
Antimicrobial resistance (AMR) is of paramount importance in the context of One Health, an integrated and unifying approach that aims to achieve a sustainable balance in the well-being of people, domestic and wild animals, plants, and their shared environments. Whenever bacteria become resistant to the therapeutic effects of antibiotics, they can cause infections that are difficult to treat effectively, increasing the risk of severe disease progression and death. Although AMR can develop naturally over time and is per se “ancient”, the excessive use of antibiotics in human and veterinary medicine over the past century has significantly accelerated its emergence and spread. Opportunistic Gram-negative enterobacteria, particularly Escherichia coli (E. coli ) and Klebsiella pneumoniae (K. pneumoniae) strains, increasingly exhibit resistance to multiple classes of clinically used antibiotics, thus presenting multidrug-resistant (MDR) phenotypes. To make matters worse, some of these strains combine multidrug resistance with high-level virulence, posing a threat to both immunocompromised and healthy individuals. Consequently, MDR E. coli and K. pneumoniae have been designated as high-risk pathogens by the World Health Organization, underscoring the urgent need for new antibiotic development.
This thesis is motivated by the fact that only a limited number of international high-risk clonal E. coli and K. pneumoniae lineages stand out across all One Health dimensions and dominate the broad pool of MDR enterobacteria. While we only know little about the underlying drivers and contributing factors impacting their occurrence, emergence, and adaptation across different ecologies, this thesis employs a diverse range of bioinformatics and phenotypic approaches to identify the key factors important for the success of these lineages, also in rather under-explored settings. It includes three main components: (i) the analysis of genomic survey data of MDR E. coli isolates from ecologies in sub-Saharan Africa, (ii) the application of functional genomics and phenotyping techniques to characterize bacterial virulence and assess its clinical relevance in a food-borne E. coli strain, and (iii) the investigation of evolutionary pathways that promote the development of resistance to a novel drug combination and exploring compensatory mechanisms in a K. pneumoniae strain. To achieve these objectives, this research integrates genomics and transcriptomics with molecular biology and functional studies encompassing a comprehensive set of in vitro and in vivo virulence and resilience assays to explore MDR bacteria in-depth.
We provide compelling evidence for the broad occurrence of successful high-risk clonal lineages in the One Health context and their circulation among clinics, wildlife, and food in international locations. In the first study, we isolated extended-spectrum β-lactamase (ESBL)-producing E. coli strains from houseflies collected from various wards at the University Teaching Hospital of Butare (Rwanda). In a follow-up study, we then examined in-depth the genomes of additional ESBL-producing E. coli from the same clinic and obtained from hospitalized patients, their caregivers, associated community members, and pets. The analyses revealed that the sample sets from this sub-Saharan African context consisted predominantly of globally recognized E. coli lineages, including sequence types (ST)131, ST167, ST410, and ST617. They play a pivotal role in the further dissemination and stabilization of AMR across diverse habitats within the One Health context. Moreover, our genomic results emphasize that these One Health-related high-risk clonal lineages exhibit the ability to successfully combine multidrug resistance with high-level bacterial virulence.
To gain a more detailed understanding of the sophisticated interplay of virulence and AMR, we developed and refined a set of in vitro and in vivo methods for virulence phenotyping. These methodologies enabled us to characterize pathogens based on crucial clinical aspects such as biofilm formation, siderophore secretion, resistance to complement-mediated killing, and their capacity to cause mortality in Galleria mellonella larvae. By using a food-borne E. coli strain from an internationally recognized high-risk clonal lineage, we verified the remarkable combination of a MDR phenotype with clinically significant virulence properties, including synthesis of curli fibers and cellulose as part of biofilm formation, extensive secretion of siderophores, resilience against complement-containing human serum and pronounced mortality in the infection model.
Nevertheless, the success of One Health-related high-risk clonal lineages does not rely solely on an “ideal” synergistic interplay between bacterial virulence and AMR. It also depends on their ability to rapidly mitigate the fitness costs associated with AMR acquisition, as these costs manifest in the form of reduced competitiveness and virulence in the absence of antibiotics. However, this is at odds with the observation of the global distribution of One Health-related high-risk clonal lineages across various One Health dimensions, even in environments with expectedly low selection pressures. To comprehensively address this, we conducted experimental evolution studies selecting for ceftazidime-avibactam-resistant mutants, which illuminated the rapid adaptations to changing environments. The adaptations and compensatory mechanisms were seemingly driven by major bacterial regulators, including the envelope stress response regulator RpoE on genomic and transcriptomic levels.
In conclusion, the results of this thesis shed light on the fundamental principles that govern the character and interplay between AMR and bacterial virulence and advance our understanding of the contributors and drivers of successful MDR international high-risk clonal lineages in the One Health context. This is also important for effective and alternative intervention strategies to prospectively further address the global threat of AMR.
Background and Objectives: Alzheimer’s disease (AD) stands as a pervasive neurodegenerative ailment of global concern, necessitating a relentless pursuit of remedies. This study aims to furnish a comprehensive exposition, delving into the intricate mechanistic actions of medicinal herbs and phytochemicals. Furthermore, we assess the potential of these compounds in inhibiting human acetylcholinesterase through molecular docking, presenting encouraging avenues for AD therapeutics. Materials and Methods: Our approach entailed a systematic exploration of phytochemicals like curcumin, gedunin, quercetin, resveratrol, nobiletin, fisetin, and berberine, targeting their capability as human acetylcholinesterase (AChE) inhibitors, leveraging the PubChem database. Diverse bioinformatics techniques were harnessed to scrutinize molecular docking, ADMET (absorption, distribution, metabolism, excretion, and toxicity), and adherence to Lipinski’s rule of five. Results: Results notably underscored the substantial binding affinities of all ligands with specific amino acid residues within AChE. Remarkably, gedunin exhibited a superior binding affinity (−8.7 kcal/mol) compared to the reference standard. Conclusions: These outcomes accentuate the potential of these seven compounds as viable candidates for oral medication in AD treatment. Notably, both resveratrol and berberine demonstrated the capacity to traverse the blood-brain barrier (BBB), signaling their aptitude for central nervous system targeting. Consequently, these seven molecules are considered orally druggable, potentially surpassing the efficacy of the conventional drug, donepezil, in managing neurodegenerative disorders.
Despite recent advances in the treatment of non-small cell lung cancer (NSCLC), acquired drug resistance to targeted therapy remains a major obstacle. Epithelial-mesenchymal transition (EMT) has been identified as a key resistance mechanism in NSCLC. Here, we investigated the mechanistic role of key EMT-regulating small non-coding microRNAs (miRNAs) in sublines of the NSCLC cell line HCC4006 adapted to afatinib, erlotinib, gefitinib, or osimertinib. The most differentially expressed miRNAs derived from extracellular vesicles were associated with EMT, and their predicted target ZEB1 was significantly overexpressed in all resistant cell lines. Transfection of a miR-205-5p mimic partially reversed EMT by inhibiting ZEB1, restoring CDH1 expression, and inhibiting migration in erlotinib-resistant cells. Gene expression of EMT-markers, transcription factors, and miRNAs were correlated during stepwise osimertinib adaptation of HCC4006 cells. Temporally relieving cells of osimertinib reversed transition trends, suggesting that the implementation of treatment pauses could provide prolonged benefits for patients. Our results provide new insights into the contribution of miRNAs to drug-resistant NSCLC harboring EGFR-activating mutations and highlight their role as potential biomarkers and therapeutic targets.
Because of the importance of gastric emptying for pharmacokinetics, numerous methods have been developed for its determination. One of the methods is the salivary tracer technique, which utilizes an ice capsule containing caffeine as a salivary tracer. Despite the ice capsule’s advantage in labeling ingested fluids with caffeine for subsequent salivary detection, its risk of premature melting before swallowing, and its complicated storage and preparation, limit its application, particularly in special populations (e.g., older people). For this reason, here, a compression-coated tablet was developed and validated against the ice capsule in a cross-over clinical trial. The two dosage forms were administered simultaneously to 12 volunteers in an upright position under fasted and fed state conditions. To distinguish the caffeine concentrations in saliva from each dosage form, regular type of caffeine (12C) was added to the tablet, while for the ice capsule 13C3 labelled caffeine was used. The salivary caffeine concentrations showed no statistically significant differences for the pharmacokinetic parameters tmax and AUC0→60 (p > 0.05). Thus, the new formulation is a useful tool for determining gastric emptying that can also be used in special populations.
Dermatophytosis, the most prevalent fungal infection, is witnessing a rising incidence annually. To address this challenge, we developed a terbinafine-loaded oil-in-water nanoemulsion (TH-NE) through the aqueous microtitration method. The formulation comprised olive oil (oil phase), Span 80 (surfactant), and propylene glycol (co-surfactant). Pseudo-phase ternary diagrams and thermodynamic studies underscored the stability of TH-NE. Employing the Box–Behnken design (BBD), we optimized TH-NE, which resulted in a remarkable particle size of 28.07 nm ± 0.5, a low polydispersity index (PDI) of 0.1922 ± 0.1, and a substantial negative zeta potential of −41.87 mV ± 1. Subsequently, TH-NE was integrated into a 1.5% carbopol matrix, yielding a nanoemulgel (TH-NEG). Texture analysis of TH-NEG demonstrated a firmness of 168.00 g, a consistency of 229.81 g/s, negative cohesiveness (−83.36 g), and a work of cohesion at −107.02 g/s. In vitro drug release studies revealed an initial burst effect followed by sustained release, with TH-NEG achieving an impressive 88% release over 48 h, outperforming TH-NE (74%) and the marketed formulation (66%). Ex vivo release studies mirrored these results, with TH-NEG (86%) and TH-NE (71%) showcasing sustained drug release in comparison to the marketed formulation (67%). Confocal microscopy illustrated that TH-NEG and TH-NE penetrated to depths of 30 µm and 25 µm, respectively, into the epidermal layer. Furthermore, dermatokinetic studies highlighted the enhanced drug penetration of TH-NEG compared to TH-NE through mouse skin. In summary, our study establishes TH-NEG as a promising carrier for terbinafine in treating dermatophytosis, offering improved drug delivery and sustained release potential.
Synthesis of Quercetin-Loaded Silver Nanoparticles and Assessing Their Anti-Bacterial Potential
(2023)
The study delves into the multifaceted potential of quercetin (Qu), a phytoconstituent found in various fruits, vegetables, and medicinal plants, in combination with silver nanoparticles (AgNPs). The research explores the synthesis and characterization of AgNPs loaded with Qu and investigates their pharmaceutical applications, particularly focusing on antibacterial properties. The study meticulously evaluates Qu’s identity, and physicochemical properties, reaffirming its suitability for pharmaceutical use. The development of Qu-loaded AgNPs demonstrates their high drug entrapment efficiency, ideal particle characteristics, and controlled drug release kinetics, suggesting enhanced therapeutic efficacy and reduced side effects. Furthermore, the research examines the antibacterial activity of Qu in different solvents, revealing distinct outcomes. Qu, both in methanol and water formulations, exhibits antibacterial activity against Escherichia coli, with the methanol formulation displaying a slightly stronger efficacy. In conclusion, this study successfully synthesizes AgNPs loaded with Qu and highlights their potential as a potent antibacterial formulation. The findings underscore the influence of solvent choice on Qu’s antibacterial properties and pave the way for further research and development in drug delivery systems and antimicrobial agents. This innovative approach holds promise for addressing microbial resistance and advancing pharmaceutical formulations for improved therapeutic outcomes.
The goal of this study was to assess the anticancer efficacy of chlorojanerin against various cancer cells. The effects of chlorojanerin on cell cytotoxicity, cell cycle arrest, and cell apoptosis were examined using MTT assay, propidium iodide staining, and FITC Annexin V assay. RT-PCR was employed to determine the expression levels of apoptosis-related genes. Furthermore, docking simulations were utilized to further elucidate the binding preferences of chlorojanerin with Bcl-2. According to MTT assay, chlorojanerin inhibited the proliferation of all tested cells in a dose-dependent manner with a promising effect against A549 lung cancer cells with an IC50 of 10 µM. Cell growth inhibition by chlorojanerin was linked with G2/M phase cell cycle arrest in A549 treated cells. Flow cytometry analysis indicated that the proliferation inhibition effect of chlorojanerin was associated with apoptosis induction in A549 cells. Remarkably, chlorojanerin altered the expression of many genes involved in apoptosis initiation. Moreover, we determined that chlorojanerin fit into the active site of Bcl-2 according to the molecular docking study. Collectively, our results demonstrate that chlorojanerin mediated an anticancer effect involving cell cycle arrest and apoptotic cell death and, therefore, could potentially serve as a therapeutic agent in lung cancer treatment.
The absorption of drugs with narrow absorption windows in the upper small intestine can be improved with a mucoadhesive drug delivery system such as enteric films. To predict the mucoadhesive behaviour in vivo, suitable in vitro or ex vivo methods can be performed. In this study, the influence of tissue storage and sampling site on the mucoadhesion of polyvinyl alcohol film to human small intestinal mucosa was investigated. Tissue from twelve human subjects was used to determine adhesion using a tensile strength method. Thawing of tissue frozen at −20 °C resulted in a significantly higher work of adhesion (p = 0.0005) when a low contact force was applied for one minute, whereas the maximum detachment force was not affected. When the contact force and time were increased, no differences were found for thawed tissue compared to fresh tissue. No change in adhesion was observed depending on the sampling location. Initial results from a comparison of adhesion to porcine and human mucosa suggest that the tissues are equivalent.
Development of Test Programs for the Biorelevant Characterization of Esophageal-Applied Dosage Forms
(2023)
In the local treatment of the esophageal mucosa, the retention time of the different dosage forms, such as tablets, films or liquids, is of high relevance for the effective treatment of diseases. Unfortunately, there are only few in vitro models describing the esophageal route of administration. To predict the behaviour of an esophageal-applied dosage form, it is necessary to simulate the site of application in a biorelevant way. The aim of this work was to develop two test setups for an esophageal peristalsis model which was described in a previous study. Different parameters such as flow rate, peristalsis, angle of inclination or mucous membrane were varied or introduced into the model. A stimulated and unstimulated modus were developed and tested with two different dosage forms. The time until the dosage form was cleared from the in vitro model was shorter with the stimulated than with the unstimulated modus. Also, esophageal-applied films had a prolonged transit time compared to a viscous syrup. The modification of the simulated esophageal surface made it possible to estimate the retention time of the dosage forms. It could be demonstrated that the residence time of a dosage form depends on different parameters affecting each other.
Humans consume snail flesh as part of their diet. To assess its nutritional value and toxicity, chemical analyses were conducted to confirm the presence of protein, total and reduced carbohydrates, fat, fatty acid composition and mineral components. Furthermore, an acute toxicity study was carried out to determine the safety of Helix aspersa Müller snail flesh. H. aspersa Müller snail flesh exhibits a high nutritional content, a good ω3/ω6 ratio and higher levels of unsaturated fatty acids. Various minerals have been found in the flesh of H. aspersa Müller. Around 76.91 kcal, or 3.84% of the energy of a daily meal of 2000 kcal, are present in 100 g of this flesh. The evaluation of the antioxidant capacity indicated that the flesh’s extracts contained a large quantity of antioxidant biomolecules. Administration of the aqueous extract of H. aspersa Müller flesh didn’t cause death in laboratory rats, indicating that the lethal dose 50 is greater than 2000 mg·kg−1 body weight. The consumption of the flesh of H. aspersa Müller is highly recommended for human consumption due to its high concentration of nutrients and essential elements, as well as unsaturated fats, and due to its safety.
The microbiome of the colon is characterized by its great diversity. This varies not only intra- but also interindividually and is influenced by endogenous and exogenous factors, such as dietary and lifestyle factors. The aim of this work was to investigate the extent to which the degradation of the drug sulfasalazine is influenced by different microbiota. Therefore, the in vitro model MimiCol3 was used, which represents the physiological conditions of the ascending colon. In addition to a representative physiological volume, the pH value, redox potential and an anaerobic atmosphere are important to provide the bacteria with the best possible growth conditions. Stool samples were taken from three healthy subjects, comparing omnivorous, vegetarian and meat-rich diets, and cultured for 24 h. However, the nutrient medium used for cultivation led to the alignment of the bacterial composition of the microbiota. The previously observed differences between the diets could not be maintained. Nevertheless, the similar degradation of sulfasalazine was observed in all microbiota studied in MimiCol3. This makes MimiCol3 a suitable in vitro model for metabolism studies in the gut microbiome.
Multidrug-resistant gram-negative pathogens such as Escherichia coli have become increasingly difficult to treat and therefore alternative treatment options are needed. Targeting virulence factors like biofilm formation could be one such option. Inhibition of biofilm-related structures like curli and cellulose formation in E. coli has been shown for different phenolic natural compounds like epigallocatechin gallate. This study demonstrates this effect for other structurally unrelated phenolics, namely octyl gallate, scutellarein and wedelolactone. To verify whether these structurally different compounds influence identical pathways of biofilm formation in E. coli a broad comparative RNA-sequencing approach was chosen with additional RT-qPCR to gain initial insights into the pathways affected at the transcriptomic level. Bioinformatical analysis of the RNA-Seq data was performed using DESeq2, BioCyc and KEGG Mapper. The comparative bioinformatics analysis on the pathways revealed that, irrespective of their structure, all compounds mainly influenced similar biological processes. These pathways included bacterial motility, chemotaxis, biofilm formation as well as metabolic processes like arginine biosynthesis and tricarboxylic acid cycle. Overall, this work provides the first insights into the potential mechanisms of action of novel phenolic biofilm inhibitors and highlights the complex regulatory processes of biofilm formation in E. coli.
For the characterization of Kv7.2/3 channel activators, several analytical methods are available that vary in effort and cost. In addition to the technically elaborate patch-clamp method, which serves as a reference method, there exist several medium to high-throughput screening methods including a rubidium efflux flame-atomic absorption spectrometry (F-AAS) assay and a commercial thallium uptake fluorescence-based assay. In this study, the general suitability of a graphite furnace atomic absorption spectrometry (GF-AAS)-based rubidium efflux assay as a screening method for Kv7.2/3 channel activators was demonstrated. With flupirtine serving as a reference compound, 16 newly synthesizedcompounds and the known Kv7.2/3 activator retigabine were first classified as either active or inactive by using the GF-AAS-based rubidium (Rb) efflux assay. Then, the results were compared with a thallium (Tl) uptake fluorescence-based fluorometric imaging plate reader (FLIPR) potassium assay. Overall, 16 of 17 compounds were classified by the GF-AAS-based assay in agreement with their channel-activating properties determined by the more expensive Tl uptake, fluorescence-based assay. Thus, the performance of the GF-AAS-based Rb assay for primary drug screening of Kv7.2/3-activating compounds was clearly demonstrated, as documented by the calculated Z’-factor of the GF-AAS-based method. Moreover, method development included optimization of the coating of the microtiter plates and the washing procedure, which extended the range of this assay to poorly adherent cells such as the HEK293 cells used in this study.
Flupirtine and retigabine were essential drugs to combat pain and epilepsy. However, the Kv7 potassium channel openers are fraught with hepatotoxicity and tissue discoloration, respectively, limiting their therapeutic value. Both adverse events are likely due to reactive metabolites arising from oxidative metabolism. Designing safer analogues lacking the structural elements leading to described side effects is an active area of current research. One of the main metabolites of flupirtine is the biologically inactive 4-fluorohippuric acid. Hitherto unexplained, the proposed metabolic pathway leading to the formation of 4-fluorohippuric acid from flupirtine is verified here. Through the use of eighteen flupirtine analogues, mechanistic details of this pathway could be elucidated. A possible connection with the in vitro hepatotoxicity of the flupirtine analogues and the levels of 4-fluorobenzoic acid formed in enzyme incubations was examined by correlation analysis. These findings provide important information for the design of new flupirtine analogues as potential drug candidates.
Marine Bacteroidetes that degrade polysaccharides contribute to carbon cycling in the ocean. Organic matter, including glycans from terrestrial plants, might enter the oceans through rivers. Whether marine bacteria degrade structurally related glycans from diverse sources including terrestrial plants and marine algae was previously unknown. We show that the marine bacterium Flavimarina sp. Hel_I_48 encodes two polysaccharide utilization loci (PULs) which degrade xylans from terrestrial plants and marine algae. Biochemical experiments revealed activity and specificity of the encoded xylanases and associated enzymes of these PULs. Proteomics indicated that these genomic regions respond to glucuronoxylans and arabinoxylans. Substrate specificities of key enzymes suggest dedicated metabolic pathways for xylan utilization. Some of the xylanases were active on different xylans with the conserved β-1,4-linked xylose main chain. Enzyme activity was consistent with growth curves showing Flavimarina sp. Hel_I_48 uses structurally different xylans. The observed abundance of related xylan-degrading enzyme repertoires in genomes of other marine Bacteroidetes indicates similar activities are common in the ocean. The here presented data show that certain marine bacteria are genetically and biochemically variable enough to access parts of structurally diverse xylans from terrestrial plants as well as from marine algal sources.
Overexpression of polo-like kinase 1 (PLK1) has been found in many different types of cancers. With its essential role in cell proliferation, PLK1 has been determined to be a broad-spectrum anti-cancer target. In this study, 3D-QSAR, molecular docking, and molecular dynamics (MD) simulations were applied on a series of novel pteridinone derivatives as PLK1 inhibitors to discover anti-cancer drug candidates. In this work, three models—CoMFA (Q² = 0.67, R² = 0.992), CoMSIA/SHE (Q² = 0.69, R² = 0.974), and CoMSIA/SEAH (Q² = 0.66, R² = 0.975)—of pteridinone derivatives were established. The three models that were established gave R²(pred) = 0.683, R²(pred) = 0.758, and R²(pred) = 0.767, respectively. Thus, the predictive abilities of the three proposed models were successfully evaluated. The relations between the different champs and activities were well-demonstrated by the contour chart of the CoMFA and CoMSIA/SEAH models. The results of molecular docking indicated that residues R136, R57, Y133, L69, L82, and Y139 were the active sites of the PLK1 protein (PDB code: 2RKU), in which the more active ligands can inhibit the enzyme of PLK1. The results of the molecular dynamic MD simulation diagram were obtained to reinforce the previous molecular docking results, which showed that both inhibitors remained stable in the active sites of the PLK1 protein (PDB code: 2RKU) for 50 ns. Finally, a check of the ADME-Tox properties of the two most active molecules showed that molecular N° 28 could represent a good drug candidate for the therapy of prostate cancer diseases.
Pentathiepins are cyclic polysulfides that exert antiproliferative and cytotoxic activity in cancer cells, induce oxidative stress and apoptosis, and potently inhibit GPx1. These properties render this class of compounds promising candidates for the development of anticancer drugs. However, the biological effects and how they intertwine to promote high cytotoxicity have not been systematically assessed throughout a panel of cancer cell lines from distinct tissues of origin. In this thesis, six novel pentathiepins were analyzed and constitute the second generation of compounds with additional properties such as fluorescence or improved water solubility to facilitate cellular testing. All compounds underwent extensive biological evaluation in 14 human cancer cell lines. These studies included investigations of the inhibitory potential with regards to GPx1 and cell proliferation, examined the cytotoxicity in human cancer cell lines, as well as the induction of oxidative stress and DNA strand breaks. Furthermore, selected hallmarks of apoptosis, ferroptosis, and autophagy were studied. Experimental approaches regarding these cellular mechanisms included observing morphological changes, detecting phosphatidyl serine exposure and caspase activity, and quantifying cleaved PARP1 and levels of LC3B II. In addition, the analysis of the cell cycle aimed to identify aberrations or arrests in cell division.
Five of the six tested pentathiepins proved to be potent inhibitors of the GPx1, while all six exerted high cytotoxic and antiproliferative activity, although to different extents. There was a clear connection observed between the potential to provoke oxidative stress and damage to DNA in the form of single- and double-strand breaks both extra- and intracellularly. Furthermore, various experiments supported apoptosis but not ferroptosis as the mechanism of cell death in four different cell lines. In particular, the externalization of PS, the detection of activated caspases, and the cleavage of PARP1 corroborated this conclusion. Additionally, indications for autophagy were found, but more investigations are required to verify the current data. The findings of this dissertation are mainly in line with the postulated mechanism of action proposed for pentathiepins and a previous publication from our group that described their biological activity. However, the influence of modulators such as oxygen and GSH on the biological effects was ambiguous and dependent on the compound. The expression profile of the cell lines concerning GPx1 and CAT did not influence the cellular response toward the treatment, whereas the cell doubling time correlated with the cytotoxicity.
As the various pentathiepins give rise to different biological responses, modulation of the biological effects depends on the distinct chemical structures fused to the sulfur ring. This may allow for future optimization of the anticancer activity of pentathiepins. An analysis of the structure-activity relationships revealed that the piperazine scaffold was associated with superior biological activity compared to the pyrrolo-pyrazine backbone. Furthermore, substituents with electron-withdrawing properties or those providing a free electron pair, such as fluorine or morpholine, were advantageous. These findings should help design and synthesize the next generation of pentathiepins, thereby expanding the library of compounds, allowing for the further deduction of structure-activity relationships and an improved understanding of their mechanism of action.
The poor aqueous solubility of many drug substances has been addressed using different solubility enhancement approaches in the pharmaceutical technology field over the last decades. In this context, advanced drug delivery systems based on lipids referred to as SNEDDS were used to overcome solubility limitations of drugs, that are often associated with a low bioavailability after oral administration. There are numerous examples in the literature for the development of L-SNEDDS, which have led to some pharmaceutical products available on the market. As L-SNEDDS development using conventional methods requires a lot of time and experimental effort, a streamlining of this procedure was aimed in the first part of the presented work.
Starting with the development of L-SNEDDS formulations for solubility enhancement of poorly-water soluble drugs, extensive solubility studies with different BCS Class II drugs were performed in various excipients to determine drugs with high solubilities in these excipients as well as to evaluate multiple excipients for their suitability to be used in L-SNEDDS formulations. Celecoxib, efavirenz and fenofibrate were selected as model drugs and a pre-selection of excipients for further development was made. In a next step, a novel screening approach for L-SNEDDS formulation development based on a customized mapping method in a special triangular mixture design was established. This customized tool for L-SNEDDS development comprised the systematic analysis of results obtained with different in vitro characterization methods such as droplet size analysis and distribution, transmittance measurement and emulsification performance assessment. Furthermore, the novel approach streamlined the procedure for L-SNEDDS development as a reduction of experimental effort and time compared to conventional methods was achieved. The most promising L-SNEDDS formulations determined via the customized screening tool approach showed high drug release of celecoxib, efavirenz as well as fenofibrate, and clearly indicated that this method was suitable for efficiently designing stable and rapidly releasing L-SNEDDS formulations incorporating poorly water-soluble drugs.
After the successful development of L-SNEDDS formulations with different drug substances using the novel screening approach, a further aspect of this work dealt with conversion of L-SNEDDS into S-SNEDDS, since a limited storage stability has been reported for many L-SNEDDS formulations. The conversion into S-SNEDDS required the determination of appropriate solid carriers with different material properties depending on the manufacturing process. As a first technological approach, adsorption to a solid carrier was investigated by adding a carrier to drug-loaded L-SNEDDS applying a defined mixing ratio resulting in a solid, particulate formulation. When performing drug release studies, S-SNEDDS based on different commercial
carrier materials revealed major limitations due to incomplete drug release. Thus, a tailor-made microparticulate carrier material based on cellulose was developed for the purpose of adsorbing L-SNEDDS and presented with superior performance compared to conventional adsorbents based on cellulose or silica. Based on the obtained results, this novel cellulose-based microparticle prepared with gum arabic as a binder was determined to be the most promising material amongst all adsorptive carriers that were investigated.
In addition to the technology approach of adsorption, another manufacturing process was considered in the course of the present work, which focused on the preparation of S-SNEDDS by means of HME. As a successful conversion of L-SNEDDS into S-SNEDDS using HME processing requires at least one additional polymeric component, a selection of marketed (co-)polymers that were frequently used in the field of solubility enhancement were evaluated for their suitability in this context. Critical process parameters and target properties of the (co-)polymers were determined, ultimately leading to the idea of developing a novel, customized polymer in order to perform the conversion step via HME in a more suitable and effective manner. In this context, a new copolymer referred to as ModE, as it disclosed a structural association with the commercially available copolymer EUDRAGIT® E PO, was developed. The novel copolymer ModE was evaluated for its suitability for different formulation technologies and showed promising results when used for S-SNEDDS and ASD formulations prepared by the HME process. Different variants of ModE in terms of Mw, Tg and PDI were synthesized via radical polymerization and it was found that the modification of Mw, Tg and PDI of the novel aminomethacrylate-based copolymer had significant effects on drug release as well as storage stability of S-SNEDDS and ASDs. The ModE copolymer type with a Mw of 173 kDa turned out to be the most suitable candidate for S-SNEDDS development using HME technology. In addition, drug-loaded S-SNEDDS based on the ModE variant 173 kDa were storage stable and presented with the highest drug release among all S-SNEDDS formulations tested.
In conclusion, a novel screening tool approach for efficient L-SNEDDS development was established in order to streamline the process for obtaining stable and rapidly releasing L-SNEDDS formulations which improved the solubility of poorly water-soluble drugs. Apart from the L-SNEDDS development process, the conversion from L-SNEDDS into S-SNEDDS was successfully performed using the technology approaches of adsorption to a solid carrier and HME processing. An improved storage stability compared to L-SNEDDS as well as high drug release were achieved for several S-SNEDDS formulations, especially for those prepared with tailor-made materials. Based on the results obtained for S-SNEDDS formulations produced via adsorption, especially in terms of drug release performance, the new cellulose-based
microparticle carriers (M-GA and M-MC) turned out to be the most suitable materials. S-SNEDDS that were manufactured via HME presented with a superior performance regardless of the incorporated drug when comparing the results of S-SNEDDS with those of the corresponding ASDs regarding drug release performance, amorphicity/crystallinity and storage stability. In this context, among all S-SNEDDS formulations prepared via HME, S-SNEDDS based on the ModE variant 173 kDa showed the best results, especially when using the drug substances celecoxib and efavirenz. Although the S-SNEDDS formulation approach is still largely unexplored, based on the research results generated in the present work, it represents a promising technology platform that should definitely be further developed in future experiments.
Klebsiella pneumoniae is a common member of the intestinal flora of vertebrates. In addition to opportunistic representatives, hypervirulent (hvKp) and antibiotic-resistant K. pneumoniae (ABR-Kp) occur. While ABR-Kp isolates often cause difficult-to-treat diseases due to limited therapeutic options, hvKp is a pathotype that can infect healthy individuals often leading to recurrent infection. Here, we investigated the clinical K. pneumoniae isolate PBIO3459 obtained from a blood sample, which showed an unusual colony morphology. By combining whole-genome and RNA sequencing with multiple in vitro and in vivo virulence-associated assays, we aimed to define the respective Klebsiella subtype and explore the unusual phenotypic appearance. We demonstrate that PBIO3459 belongs to sequence type (ST)20 and carries no acquired resistance genes, consistent with phenotypic susceptibility tests. In addition, the isolate showed low-level virulence, both at genetic and phenotypic levels. We thus suggest that PBIO3459 is an opportunistic (commensal) K. pneumoniae isolate. Genomic comparison of PBIO3459 with closely related ABR-Kp ST20 isolates revealed that they differed only in resistance genes. Finally, the unusual colony morphology was mainly associated with carbohydrate and amino acid transport and metabolism. In conclusion, our study reveals the characteristics of a Klebsiella sepsis isolate and suggests that opportunistic representatives likely acquire and accumulate antibiotic resistances that subsequently enable their emergence as ABR-Kp pathogens.