Refine
Has Fulltext
- yes (34)
Is part of the Bibliography
- yes (34)
Year of publication
Document Type
- Doctoral Thesis (34)
Keywords
- Bioverfügbarkeit (7)
- Löslichkeit (5)
- Arzneimittel (4)
- Ionic Liquids (4)
- Tissue Engineering (4)
- Extrazelluläre Matrix (3)
- Formulierungsentwicklung (3)
- Interleukin 4 (3)
- Kontrollierte Wirkstofffreisetzung (3)
- Solubilisation (3)
Institute
- Institut für Pharmazie und Lebensmittelchemie (29)
- Graduate School of Life Sciences (4)
- Abteilung für Funktionswerkstoffe der Medizin und der Zahnheilkunde (1)
- Fakultät für Chemie und Pharmazie (1)
- Institut für Molekulare Infektionsbiologie (1)
- Institut für Organische Chemie (1)
- Julius-von-Sachs-Institut für Biowissenschaften (1)
Sonstige beteiligte Institutionen
This thesis aimed at searching for new effective agents against Multidrug-Resistant Enterobacteriaceae. This is necessitated by the urgent need for new and innovative antibacterial agents addressing the critical priority pathogens prescribed by the World Health Organization (WHO). Among the available means for antibiotics discovery and development, nature has long remained a proven, innovative, and highly reliable gateway to successful antibacterial agents. Nevertheless, numerous challenges surrounding this valuable source of antibiotics among other drugs are limiting the complete realization of its potential. These include the availability of good quality data on the highly potential natural sources, limitations in methods to prepare and screen crude extracts, bottlenecks in reproducing biological potentials observed in natural sources, as well as hurdles in isolation, purification, and characterization of natural compounds with diverse structural complexities.
Through an extensive review of the literature, it was possible to prepare libraries of plant species and phytochemicals with reported high potentials against Escherichia coli and Klebsiella pneumnoniae. The libraries were profiled to highlight the existing patterns and relationships between the reported antibacterial activities and studied plants’ families and parts, the type of the extracting solvent, as well as phytochemicals’ classes, drug-likeness and selected parameters for enhanced accumulation within the Gram-negative bacteria. In addition, motivations, objectives, the role of traditional practices and other crucial experimental aspects in the screening of plant extracts for antibacterial activities were identified and discussed.
Based on the implemented strict inclusion criteria, the created libraries grant speedy access to well-evaluated plant species and phytochemicals with potential antibacterial activities. This way, further studies in yet unexplored directions can be pursued from the indicated or related species and compounds. Moreover, the availability of compound libraries focusing on related bacterial species serves a great role in the ongoing efforts to develop the rules of antibiotics penetrability and accumulation, particularly among Gram-negative bacteria. Here, in addition to hunting for potential scaffolds from such libraries, detailed evaluations of large pool compounds with related antibacterial potential can grant a better understanding of structural features crucial for their penetration and accumulation. Based on the scarcity of compounds with broad structural diversity and activity against Gram-negative bacteria, the creation and updating of such libraries remain a laborious but important undertaking.
A Pressurized Microwave Assisted Extraction (PMAE) method over a short duration and low-temperature conditions was developed and compared to the conventional cold maceration over a prolonged duration. This method aimed at addressing the key challenges associated with conventional extraction methods which require long extraction durations, and use more energy and solvents, in addition to larger quantities of plant materials. Furthermore, the method was intended to replace the common use of high temperatures in most of the current MAE applications. Interestingly, the yields of 16 of 18 plant samples under PMAE over 30 minutes were found to be within 91–139% of those obtained from the 24h extraction by maceration. Additionally, different levels of selectivity were observed upon an analytical comparison of the extracts obtained from the two methods. Although each method indicated selective extraction of higher quantities or additional types of certain phytochemicals, a slightly larger number of additional compounds were observed under maceration. The use of this method allows efficient extraction of a large number of samples while sparing heat-sensitive compounds and minimizing chances for cross-reactions between phytochemicals.
Moreover, findings from another investigation highlighted the low likelihood of reproducing antibacterial activities previously reported among various plant species, identified the key drivers of poor reproducibility, and proposed possible measures to mitigate the challenge. The majority of extracts showed no activities up to the highest tested concentration of 1024 µg/mL. In the case of identical plant species, some activities were observed only in 15% of the extracts, in which the Minimum Inhibitory Concentrations (MICs) were 4 – 16-fold higher than those in previous reports. Evaluation of related plant species indicated better outcomes, whereby about 18% of the extracts showed activities in a range of 128–512 μg/mL, some of the activities being superior to those previously reported in related species.
Furthermore, solubilizing plant crude extracts during the preparation of test solutions for Antibacterial Susceptibility Testing (AST) assays was outlined as a key challenge. In trying to address this challenge, some studies have used bacteria-toxic solvents or generally unacceptable concentrations of common solubilizing agents. Both approaches are liable to give false positive results. In line with this challenge, this study has underscored the suitability of acetone in the solubilization of crude plant extracts. Using acetone, better solubility profiles of crude plant extracts were observed compared to dimethyl sulfoxide (DMSO) at up to 10 %v/v. Based on lacking toxicity against many bacteria species at up to 25 %v/v, its use in the solubilization of poorly water-soluble extracts, particularly those from less polar solvents is advocated.
In a subsequent study, four galloylglucoses were isolated from the leaves of Paeonia officinalis L., whereby the isolation of three of them from this source was reported for the first time. The isolation and characterization of these compounds were driven by the crucial need to continually fill the pre-clinical antibiotics pipeline using all available means. Application of the bioautography-guided isolation and a matrix of extractive, chromatographic, spectroscopic, and spectrometric techniques enabled the isolation of the compounds at high purity levels and the ascertainment of their chemical structures.
Further, the compounds exhibited the Minimum Inhibitory Concentrations (MIC) in a range of 2–256 µg/mL against Multidrug-Resistant (MDR) strains of E. coli and K. pneumonia exhibiting diverse MDR phenotypes. In that, the antibacterial activities of three of the isolated compounds were reported for the first time. The observed in vitro activities of the compounds resonated with their in vivo potentials as determined using the Galleria mellonella larvae model. Additionally, the susceptibility of the MDR bacteria to the galloylglucoses was noted to vary depending on the nature of the resistance enzymes expressed by the MDR bacteria. In that, the bacteria expressing enzymes with higher content of aromatic amino acids and zero or positive net charges were generally more susceptible. Following these findings, a plausible hypothesis for the observed patterns was put forward.
The generally challenging pharmacokinetic properties of galloylglucoses limit their further development into therapeutic agents. However, the compounds can replace or reduce the use of antibiotics in livestock keeping as well as in the treatment of septic wounds and topical or oral cavity infections, among other potential uses.
Using nature-inspired approaches, a series of glucovanillin derivatives were prepared following feasible synthetic pathways which in most cases ensured good yields and high purity levels. Some of the prepared compounds showed MIC values in a range of 128 – 512 μg/mL against susceptible and MDR strains of Klebsiella pneumoniae, Methicillin-Resistant Staphylococcus aureus (MRSA) and Vancomycin-Resistant Enterococcus faecium (VRE). These findings emphasize the previously reported essence of small molecular size, the presence of protonatable amino groups and halogen atoms, as well as an amphiphilic character, as crucial features for potential antibacterial agents.
Due to the experienced limited success in the search for new antibacterial agents using purely synthetic means, pursuing semi-synthetic approaches as employed in this study are highly encouraged. This way, it is possible to explore broader chemical spaces around natural scaffolds while addressing their inherent limitations such as solubility, toxicity, and poor pharmacokinetic profiles.
Serum half-life elongation as well as the immobilization of small proteins like cytokines is still one of the key challenges for biologics. This accounts also for cytokines, which often have a molecular weight between 5 and 40 kDa and are therefore prone to elimination by renal filtration and sinusoidal lining cells. To solve this problem biologics are often conjugated to poly(ethylene glycol) (PEG), which is the gold standard for the so called PEGylation. PEG is a synthetic, non-biodegradable polymer for increasing the hydrodynamic radius of the conjugated protein to modulate their pharmacokinetic performance and prolong their therapeutic outcome. Though the benefits of PEGylation are significant, they also come with a prize, which is a loss in bioactivity due to steric hindrance and most often the usage of heterogeneous bioconjugation chemistries. While PEG is a safe excipient in most cases, an increasing number of PEG related side-effects, such as immunological responses like hypersensitivity and accelerated blood clearance upon repetitive exposure occur, which highlights the need for PEG alternative polymers, that can replace PEG in such cases.
Another promising method to significantly prolong the residence time of biologics is to immobilize them at a desired location. To achieve this, the transglutaminase (TG) Factor XIIIa (FXIIIa), which is an important human enzyme during blood coagulation can be used. FXIIIa can recognize specific peptide sequences that contain a lysine as substrates and link them covalently to another peptide sequence, that contains a glutamine, forming an isopeptide bond. This mechanism can be used to link modified proteins, which have a N- or C-terminal incorporated signal peptide by mutation, to the extracellular matrix (ECM) of tissues.
Additionally, both above-described methods can be combined. By artificially introducing a TG recognition sequence, it is possible to attach an azide group containing peptide site-specifically to the TG, recognition sequence. This allows the creation of a site-selective reactive site at the proteins N- or C-terminus, which can then be targeted by cyclooctyne functionalized polymers, just like amber codon functionalized proteins.
This thesis has focused on the two cytokines human Interferon-α2a (IFN-α2a) and human, as well as murine Interleukin-4 (IL-4) as model proteins to investigate the above-described challenges. IFN-α2a has been chosen as a model protein because it is an approved drug since 1986 in systemic applications against some viral infections, as well as several types of cancer. Furthermore, IFN-α2 is also approved in three PEGylated forms, which have different molecular weights and use different conjugation techniques for polymer attachment. This turns it into an ideal candidate to compare new polymers against the gold standard PEG. Interleukin-4 (IL-4) has been chosen as the second model protein due to its similar size and biopotency. This allows to compare found trends from IFN-α2a with another bioconjugate platform and distinguish between IFN-α2a specific, or general trends. Furthermore, IL-4 is a promising candidate for clinical applications as it is a potent anti-inflammatory protein, which polarizes macrophages from the pro-inflammatory M1 state into the anti-inflammatory M2 state.
Most medicines are taken orally. To enter the systemic circulation, they dissolve in the intestinal fluid, cross the epithelial barrier, and pass through the liver. Intestinal absorption is driven by the unique features of the gastrointestinal tract, including the bile colloids formed in the lumen and the mucus layer covering the intestinal epithelium. Neglecting this multifaceted environment can lead to poor drug development decisions, especially for poorly water-soluble drugs that interact with bile and mucus. However, there is a lack of a rationale nexus of molecular interactions between oral medicines and gastrointestinal components with drug bioavailability. Against this background, this thesis aims to develop biopharmaceutical strategies to optimize the presentation of oral therapeutics to the intestinal epithelial barrier.
In Chapter 1, the dynamics of bile colloids upon solubilization of the poorly-water soluble drug Perphenazine was studied. Perphenazine impacted molecular arrangement, structure, binding thermodynamics, and induced a morphological transition from vesicles to worm-like micelles. Despite these dynamics, the bile colloids ensured stable relative amounts of free drug substance. The chapter was published in Langmuir.
Chapter 2 examined the impact of pharmaceutical polymeric excipients on bile-mediated drug solubilization. Perphenazine and Imatinib were introduced as model compounds interacting with bile, whereas Metoprolol did not. Some polymers altered the arrangement and geometry of bile colloids, thereby affecting the molecularly soluble amount of those drugs interacting with bile. These insights into the bile-drug-excipient interplay provide a blueprint to optimizing formulations leveraging bile solubilization. The chapter was published in Journal of Controlled Release.
Chapter 3 deals with the impact of bile on porcine intestinal mucus. Mucus exposed to bile solution changed transiently, it stiffened, and the overall diffusion rate increased. The bile-induced changes eased the transport of the bile-interacting drug substance Fluphenazine, whereas Metoprolol was unaffected. This dichotomous pattern was linked to bioavailability in rats and generalized based on two previously published data sets. The outcomes point to a bile-mucus interaction relevant to drug delivery. The chapter is submitted.
The Appendix provides a guide for biopharmaceutical characterization of drug substances by nuclear magnetic resonance spectroscopy aiming at establishing a predictive algorithm.
In summary, this thesis deciphers bile-driven mechanisms shaping intestinal drug absorption. Based on these molecular insights, pharmaceuticals can be developed along a biopharmaceutical optimization, ultimately leading to better oral drugs of tomorrow.
Poor or variable oral bioavailability is of major concern regarding safety and efficacy for the treatment of patients with poorly water-soluble drugs (PWSDs). The problem statement of this work involves a pharmaceutical development perspective, the physicochemical basis of the absorption process and physiological / biopharmaceutical aspects. A methodology was developed aiming at closing the gap between drug liberation and dissolution on the one hand and the appearance of drug in the blood on the other. Considering what is out of control from a formulation development perspective, a clear differentiation between bioavailability and bioaccessibility was necessary. Focusing on the absorption process, bioaccessibility of a model compound, a poorly soluble but well permeable weak base, was characterized by means of flux across artificial biomimetic membranes. Such setups can be considered to reasonably mimic relevant oral absorption resistances in vitro in terms of diffusion through an unstirred water layer (UWL) and a lipidic barrier. Mechanistic understanding of the driving force for permeation was gained by differentiating drug species and subsequently linking them to the observed transfer rates using a bioaccessibility concept. The three key species that need to be differentiated are molecularly dissolved drug, drug associated in solution with other components (liquid reservoir) and undissolved drug (solid reservoir). An innovative approach to differentiate molecularly dissolved drug from the liquid reservoir using ultracentrifugation in combination with dynamic light scattering as control is presented. A guidance for rational formulation development of PWSDs is elaborated based on the employed model compound. It is structured into five guiding questions to help drug formulation scientists in selecting drug form, excipients and eventually the formulation principle. Overall, the relevance but also limitations of characterizing bioaccessibility were outlined with respect to practical application e.g. in early drug formulation development.