@phdthesis{KraehenbuehlAmstalden2018,
  author    = {Kr{\"a}henb{\"u}hl Amstalden, Maria Cecilia},
  title     = {Development of a bacterial responsive antibiotic release system},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163386},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {A major problem regarding public health is the emergence of antibiotic resistant bacterial strains, especially methicillin resistant Staphylococcus aureus (MRSA). This is mainly attributed to the unnecessary overuse of antimicrobial drugs by patients; however, one aspect that is often neglected is their untargeted mechanism of action, affecting not only the infection itself but also commensal bacteria which are often opportunistic pathogens causing many diseases as well. Therefore, our goal was to develop a bioresponsive antibiotic delivery system triggered by virulence factors. The designed system is comprised of a polymer to enhance its pharmacokinetic profile, a peptide cleavable linker, and the antibiotic agent itself. The bacterial protease aureolysin which is expressed by S. aureus during infections would cleave the linker and partially release the antibiotic which would be still attached to a remaining tetrapeptide. These would be cleaved by a group of proteases naturally present in plasma called aminopeptidases, finally releasing the compound. In the first part of this project, we searched for a suitable sequence to serve as a cleavable linker. It should be sensitive towards the target bacterial protease but not be cleaved by any human enzymes to guarantee the specificity of the system. Therefore, we synthesized three peptide sequences via Solid Phase Peptide Synthesis and incubated them with aureolysin as well as with many human matrix Metalloproteases. The analysis and quantification of enzymatic activity was monitored chromatographically (RP-HPLC). The plasminogen originated sequence was chosen since it was not sensitive towards MMPs, but cleaved by aureolysin. In the second part, we tried to incorporate the chosen peptide sequences as crosslinkers in hydrogel formulations. The purpose was to physically incorporate the antibiotic within the hydrogel, which would be released by the cleavage of those sequences and the consequent loosening the hydrogel net. For that purpose we used a commercially available hydrogel kit with a PVA matrix modified with maleimide, which allows a conjugation reaction with thiol functionalized crosslinkers. Three fluorophores were chosen to serve as antibiotic models and a diffusion assay was performed. Only the glomerular structured Green Fluorescent Protein (GFP) presented a low diffusion rate, thus the aureolysin release assays were performed only using this prototype. Assays showed that with a low hydrogel polymer concentration, the fluorophore either quickly diffused into the medium or was not released at all. The physical incorporation of the antibiotic within the hydrogel pores was therefore abolished as a suitable release approach. For a second attempt, we covalently bound a fluorophore to the linker, which was conjugated to the hydrogel matrix. The incubation with aureolysin and subsequent RP-HPLC analysis showed a peak with the same retention time correspondent to the fragment product after cleavage of the free linker. This is a proof that the concept of linking the peptide sequence to the antibiotic is a promising strategy for its bioresponsive release. Within the third part of this study, we analyzed the degradation of the resulted fragment after aureolysin activity and subsequent full release of the antibiotic by human aminopeptidases. We determined the concentration of those enzymes in human plasma and synthesized the fragment by conjugating the tetrapeptide sequence to aminofluorescein via EDC/NHS reaction. By incubating the construct with the lowest aminopeptidase concentration measured in plasma, the fluorophore was completely released within two hours, showing the efficacy of these enzymes as bioresponsive agents. The last part was the construction of the PEGylated linker-antibiotic. For this purpose we chose the tetracycline like antibiotic chelocardin (CHD) as our prototype. The conjugation of the linker- CHD to the polymer was performed by copper free click chemistry. The cleavage rate of the linker by aureolysin was very similar to the one obtained for the free peptide, indicating that the PEGylation does not interfere on the enzymatic activity. However, by trying to increase the loading ratio of chelocardin onto the polymer, we observed a very low cleavage rate for the system, indicating the formation of aggregates by those constructs. The designed system has proved to be a smart strategy for the delivery on demand of antibiotics in which the drug is only released by the presence of S. aureus during their virulent state.},
  subject      = {Arzneimittelforschung},
  language  = {en}
}
@phdthesis{Dolles2018,
  author    = {Dolles, Dominik},
  title     = {Development of Hybrid GPCR Ligands: Photochromic and Butyrylcholinesterase Inhibiting Human Cannabinoid Receptor 2 Agonists},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163445},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {While life expectancy increases worldwide, treatment of neurodegenerative diseases such as AD becomes a major task for industrial and academic research. Currently, a treatment of AD is only symptomatical and limited to an early stage of the disease by inhibiting AChE. A cure for AD might even seem far away. A rethinking of other possible targets is therefore necessary. Addressing targets that can influence AD even at later stages might be the key. Even if it is not possible to find a cure for AD, it is of great value for AD patients by providing an effective medication. The suffering of patients and their families might be relieved and remaining years may be spent with less symptoms and restrictions. It was shown that a combination of hCB2R agonist and BChE inhibitor might exactly be a promising approach to combat AD. In the previous chapters, a first investigation of dual-acting compounds that address both hCB2R and BChE was illustrated (figure 6.1). A set of over 30 compounds was obtained by applying SARs from BChE inhibitors to a hCB2R selective agonist developed by AstraZeneca. In a first in vitro evaluation compounds showed selectivity over hCB1R and AChE. Further investigations could also prove agonism and showed that unwanted off-target affinity to hMOP receptor could be designed out. The development of a homology model for hCB2R (based on a novel hCB1R crystal) could further elucidate the mode of action of the ligand binding. Lastly, first in vivo studies showed a beneficial effect of selected dual-acting compounds regarding memory and cognition. Since these first in vivo studies mainly aim for an inhibition of the BChE, it should be the aim of upcoming projects to proof the relevance of hCB2R agonism in vivo as well. In addition, pharmacokinetic as well as solubility studies may help to complete the overall picture. Currently, hybrid-based dual-acting hCB2R agonists and selective BChE inhibitors are under investigation in our lab. First in vitro evaluations showed improved BChE inhibition and selectivity over AChE compared to tacrine.78 Future in vitro and in vivo studies will clarify their usage as drug molecules with regard to hepatotoxicity and blood-brain barrier penetration. Since the role of hCB2R is not yet completely elucidated, the use of photochromic toolcompounds becomes an area of interest. These tool-compounds (and their biological effect) can be triggered upon irradiation with light and thus help to investigate time scales and ligand binding. A set of 5-azobenzene benzimidazoles was developed and synthesized. In radioligand binding studies, affinity towards hCB2R could be increased upon irradiation with UV-light (figure 6.2). This makes the investigated compounds the first GPCR ligands that can be activated upon irradiation (not vice versa). The aim of upcoming research will be the triggering of a certain intrinsic activity by an "efficacy-switch". For this purpose, several attempts are currently under investigation: an introduction of an azobenzene moiety at the 2-position of the benzimidazole core already led to a slight difference in efficacy upon irradiation with UV light. Another approach going on in our lab is the development of hCB1R switches based on the selective hCB1R inverse agonist rimonabant. First in vitro results are not yet available (figure 6.3).},
  subject      = {Ligand <Biochemie>},
  language  = {en}
}
@phdthesis{SchuesslergebHecht2018,
  author    = {Sch{\"u}ßler [geb. Hecht], Nina Kristin Petra},
  title     = {Novel formulation principles for bioavailability enhancement of poorly water-soluble and poorly permeable drugs},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162766},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Since four decades, high-throughput screenings have been conducted in drug discovery, fuelling the identification of potential new drug candidates. This approach, however, often promotes the detection of compounds with undesired physico-chemical properties like poor aqueous solubility or low membrane permeability. Indeed, dissolution and absorption of a drug are prerequisites for systemic exposure and therapeutic effects. Therefore, innovative strategies to optimize unfavourable performance of new drug candidates are in great demand in order to increase drug concentrations at the site of action whilst simultaneously reducing drug variability. In chapter I of this research work, hydrophobic ion pairing (HIP) is discussed as a promising strategy to improve the bioavailability of BCS class III compounds, which have high aqueous solubility and low permeability. The review points out the limitations of poorly absorbable drugs and details the approach of pairing these APIs with hydrophobic counterions. Apart from the motivation to tailor physico-chemical, biopharmaceutical and toxicological properties of BCS class III compounds, the hydrophobic ion pairing facilitates their formulation into drug delivery systems. Besides advantageous effects, disadvantages of the ion pair formation, such as the decreased aqueous solubility of the ions pair, are critically outlined. Finally, the review covers an overview of non-invasive administration routes permitted after ion pair formation, including oral/enteral, buccal, nasal, ocular and transdermal drug administration. Overall, the HIP approach offers substantial benefits regarding the bioavailability enhancement of BCS class III compounds. Chapter II concerns GHQ168 developed by Holzgrabe et al., a BCS class II compound characterized by low aqueous solubility and high permeability. GHQ168 was developed for the treatment of human African trypanosomiasis (HAT), a tropical disease for which novel active compounds are urgently needed. This lead compound was found to be very active against trypanosoma brucei brucei and trypanosoma brucei rhodesiense in cell culture assays, however, the low aqueous solubility prevented further preclinical development. To target this drawback, two different approaches were selected, including (I) the chemical modification and (II) the spray drying of GHQ168. The newly synthesized set of derivatives as well as the spray dried GHQ168 were subjected to a physico-chemical and microbiological characterization. It turned out that both approaches successfully improved aqueous solubility, however, for the derivatives of GHQ168 at the expense of activity. Furthermore, the pharmacokinetic parameters of GHQ168 and of the most active derivatives, GHQ242 and GHQ243, were evaluated. Elimination half-lives between 1.5 to 3.5 h after intraperitoneal administration and modest to strong serum albumin binding for GHQ243 (45\%) and GHQ168 (80\%) and very high binding (> 99\%) for GHQ242 were detected. The spray dried formulation of GHQ168, as well as GHQ242 and GHQ243 were investigated in two in vivo studies in mice infected with t. b. rhodesiense (STIB900), referred to as (I) stringent model and (II) early-treatment model. In the stringent model (2 applications/day on day 3-6 after infection) the mean survival duration (MSD) of mice treated with spray dried GHQ168 exceeded the MSD of the untreated control group (17 days versus 9 days), a difference that was statistically significant. In contrast, no statistical difference was observed for GHQ242 (14 days) and GHQ243 (12 days). GHQ168 was further assessed in the early-treatment model (2 applications/day on day 1-4 after infection) and again a statistically significant improvement of MSD (32 days (end of observation period) versus 7 days) was observed. Finally, exciting antitrypanosomal efficacy for the spray dried formulation of GHQ168 was demonstrated. NADPH oxidases (NOX) were found to be the main source of endothelial reactive oxygen species (ROS) formation. Chapter III reports on the formulation studies on triazolopyrimidine derivatives from the VAS library, a set of NADPH oxidase inhibitors. These were developed for the treatment of elevated ROS levels, which contribute to the development of cardiovascular diseases. Although in vitro results from numerous studies indicated promising efficacy and selectivity for the VAS-compounds, the low water solubility impeded the in vivo translation and further preclinical development. For this reason, three derivatives, VAS2870, VAS3947, and VAS4024 were physico-chemically characterized and VAS3947, the most soluble compound, was selected for further formulation studies. These approaches included (I) spray drying, (II) microemulsification and (III) complexation with cyclodextrins in order to develop formulations for oral and parenteral application. Solubility improvement of VAS3947 was successfully demonstrated for all preparations as expressed by supersaturation ratios in comparison to the solubility of the unformulated compound. For seven spray dried formulations, the ratio ranged from 3-9, and the ratio for four microemulsions was 8-19 after 120 min, respectively. The six cyclodextrin formulations achieved the highest supersaturation ratio between 3 and 174 after 20 hours. NMR measurements elucidated the inclusion of VAS3947 within the CD's cavity as well as the interaction with its outer surface. Ultimately, NOX inhibitors were opened for oral and parenteral administration for the first time. After successful solubility improvement of VAS3947, further investigations towards in vivo studies were conducted including stability studies with a focus on stability in solution and in plasma as presented in chapter IV. Furthermore, permeability and cytotoxicity assays were performed for the first time. It turned out that VAS3947 was instable in buffer and when exposed to light. Moreover, the compound showed decomposition in the presence of mouse microsomes and in human plasma. The VAS compounds contain an oxazol moiety linked to the triazolopyrimidine skeleton via a thioether. This structural element is responsible for the efficacy of the compound class, however it is susceptible to hydrolysis and to further degradation reactions. Moreover, VAS3947 harmed membrane integrity in the cell permeability assays and cytotoxicity investigations in HEK-293 and HEP-G2 cells revealed IC50 values in the same concentration range as reported for efficacy assays. Summarized, it was demonstrated that substances from the VAS library were no appropriate model compounds for ROS investigations nor suitable candidates for further preclinical development.},
  subject      = {L{\"o}slichkeit},
  language  = {en}
}
@phdthesis{Jones2018,
  author    = {Jones, Gabriel},
  title     = {Bioinspired FGF-2 delivery for pharmaceutical application},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-153179},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {In resent years the rate of biologics (proteins, cytokines and growth-factors) as newly registered drugs has steadily risen. The greatest challenge for pharmaceutical biologics poses its arrival at the desired target location due to e.g. proteolytic and pH dependent degradation, plasma protein binding, insolubility etc. Therefore, advanced drug delivery systems, where biologics are site directed immobilized to carriers mimicking endogenous storage sites such as the extra cellular matrix can enormously assist the application and consequently the release of exogenous administered pharmaceutical biologics. We have resorted to the fibroblast growth factor 2/ heparansulfate/ fibroblast growth factor bindingprotein 1 system as a model. Phase I deals with the selection and subcloning of a wild type murine FGF-2 construct into the bacterial pHis-Trx vector system for high yields of expression and fast, feasible purification measurements. This first step enables the provision of mFGF-2, which plays a pivotal part as a growth factor in the wound healing process as well as the vascularization of tumors, for future investigations. Therefore, the correct expression of mFGF-2 was monitored via MALDI-MS and SDS-PAGE, whereas the proper folding of the tertiary beta-trefoil structure was assessed by fluorescence spectroscopy. The MTT assay allowed us to ensure that the bioactivity was comparable to sourced FGF-2. In the last step, the purity; a requirement for future binding- and protein-protein interaction assays was monitored chromatographically (RP-HPLC). In addition, a formulation for freeze-drying was developed to ensure protein stability and integrity over a period of 60 days. Altogether, the bacterial expression and purification proved to be suitable, leading to bioactive and stable production of mFGF-2. In Phase II the expression, purification and characterization of FGFBP1, as the other key partner in the FGF-2/ HS/ FGFBP1 system is detailed. As FGFBP1 exhibits a complex tertiary structure, comprised of five highly conserved disulfide bonds and presumably multiple glycosylation sites, a eukaryotic expression was used. Human embryonic kidney cells (HEK 293F) as suspension cells were transiently transfected with DNA-PEI complexes, leading to expression of Fc-tagged murine FGFBP1. Different PEI to DNA ratios and expression durations were investigated for optimal expression yields, which were confirmed by western blot analysis and SDS-PAGE. LC-MS/MS analysis of trypsin and elastase digested FGFBP1 gave first insights of the three O-glycosylation sites. Furthermore, the binding protein was modified by inserting a His6-tag between the Fc-tag (for purification) and the binding protein itself to enable later complexation with radioactive 99mTc as radio ligand to track bio distribution of administered FGFBP1 in mice. Overall, expression, purification and characterization of mFGFBP1 variants were successful with a minor draw back of instability of the tag free binding protein. Combining the insights and results of expressed FGF-2 as well as FGFBP1 directed us to the investigation of the interaction of each partner in the FGF-2/ HS/ FGFBP1 system as Phase III. Thermodynamic behavior of FGF-2 and low molecular weight heparin (enoxaparin), as a surrogate for HS, under physiological conditions (pH 7.4) and pathophysiological conditions, similar to hypoxic, tumorous conditions (acidic pH) were monitored by means of isothermal titration calorimetry. Buffer types, as well as the pH influences binding parameters such as stoichiometry (n), enthalpy (ΔH) and to some extent the dissociation constant (KD). These findings paved the way for kinetic binding investigations, which were performed by surface plasmon resonance assays. For the first time the KD of full length FGFBP1 and FGF-2 was measured. Furthermore the binding behavior of FGF-2 to FGFBP1 in the presence of various heparin concentrations suggest a kinetic driven release of bound FGF-2 by its chaperone FGFBP1. Having gathered multiple data on the FGF-2 /HS /FGFBP1 system mainly in solution, our next step in Phase IV was the development of a test system for immobilized proteins. With the necessity to better understand and monitor the cellular effects of immobilized growth factors, we decorated glass slides in a site-specific manner with an RGD-peptide for adhesion of cells and via the copper(I)-catalyzed-azide-alkyne cycloaddition (CuAAC) a fluorescent dye (a precursor for modified proteins for click chemistry). Human osteosarcoma cells were able to grow an the slides and the fluorescence dye was immobilized in a biocompatible way allowing future thorough bioactivity assay such as MTT-assays and phospho-ERK-assays of immobilized growth factors.},
  subject      = {Fibroblastenwachstumsfaktor},
  language  = {en}
}
@phdthesis{HebronMwalwisi2018,
  author    = {Hebron Mwalwisi, Yonah},
  title     = {Assessment of Counterfeit and Substandard Antimalarial Medicines using High Performance Thin Layer Chromatography and High Performance Liquid Chromatography},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145821},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Although the prevalence of substandard and counterfeit pharmaceutical products is a global problem, it is more critical in resource-constrained countries. The national medicines regulatory authorities (MNRA) in these countries have limited resources to cater for regular quality surveillance programmes aimed at ensuring that medicines in circulation are of acceptable quality. Among the reasons explained to hinder the implementation of these strategies is that compendial monographs are too complicated and require expensive infrastructures in terms of environment, equipment and consumables. In this study it was therefore aimed at developing simple, precise, and robust HPLC and HPTLC methods utilizing inexpensive, readily available chemicals (methanol and simple buffers) that can determine the APIs, other API than declared one, and which are capable of impurity profiling. As an outcome of this study, three isocratic and robust HPLC and two HPTLC methods for sulfadoxine, sulfalene, pyrimethamine, primaquine, artesunate, as well as amodiaquine have been developed and validated. All HPLC methods are operated using an isocratic elution mode which means they can be implemented even with a single pump HPLC system and standard C18 columns. The densitometric sulfadoxine/sulfalene and pyrimethamine method utilizes standard TLC plates as well as inexpensive, readily available and safe chemicals (toluene, methanol, and ethyl acetate), while that for artesunate and amodiaquine requires HPTLC plates as well as triethylamine and acetonitrile due to challenges associated with the analysis of amodiaquine and poorly the detectable artesunate. These HPTLC methods can be implemented as alternative to those requiring HPLC equipment e.g. in countries that already have acquired densitometer equipment. It is understood that HPTLC methods are less sensitive, precise and accurate when compared to HPLC methods, but this hindrance can easily be addressed by sending representative samples to third party quality control laboratories where the analytical results are verified using compendial HPLC methods on a regular basis. It is therefore anticipated that the implementation of these methods will not only address the problem of limited resources required for medicines quality control but also increase the number of monitored targeted antimalarial products as well as the number of resource- constrained countries participating in quality monitoring campaigns. Moreover, the experiences and skills acquired within this work will be applied to other API groups, e. g. antibiotics, afterwards.},
  subject      = {Instrumentelle Analytik},
  language  = {en}
}
@phdthesis{Skaf2018,
  author    = {Skaf, Joseph},
  title     = {Antileishmanial and antitrypanosomal compounds from \(Achillea\) \(fragrantissima\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-167841},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {This PhD thesis is dealing with the bioassay-guided fractionation of a dichloromethane extract of the aerial parts of Achillea fragrantissima with the aim of isolation and structure isolation of the antileishmanial and/or antitrypanosomal principles in the plant.},
  subject      = {Schafgarbe <Gattung>},
  language  = {en}
}
@phdthesis{Scheffler2018,
  author    = {Scheffler, Anne},
  title     = {Entwicklung und Charakterisierung des RMCA f{\"u}r \(Rattus\) \(norvegicus\) in nukle{\"a}rer und mitochondrialer DNA},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-169880},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Mutationstests werden in vitro und in vivo durchgef{\"u}hrt. Insbesondere die ph{\"a}notypselektiven Mutationstests sind meist beschr{\"a}nkt auf die Detektion von Mutationen im Exon und gegebenenfalls in Promotorregionen. Um zun{\"a}chst die Datenlage zu den {\"u}blicherweise verwendeten in vitro Mutationstests zu erweitern und somit eine Bewertung der zu untersuchenden Substanz zu erleichtern, sollte eine Methode zur Erfassung des Mutationsspektrums etabliert und im Rahmen der Untersuchung des mutagenen Potentials des Lebensmittelinhaltsstoffes Irilon angewendet werden. Es wurde eine Methode entwickelt, welche die Sequenzierung eines jeden einzelnen im Hypoxanthin-Guanin-Phosphoribosyltransferase-Test enstandenen 6-Thioguanin-resistenten Mutanten erlaubt und somit auch R{\"u}ckschl{\"u}sse auf Mechanismen der Mutationsentstehung zul{\"a}sst. Im Rahmen der Untersuchung zum mutagenen Potential des Lebensmittelinhaltsstoffes Irilon, wurde zwar kein Unterschied in der Mutantenfrequenz, jedoch sehr wohl ein mit steigenden Deletionen und sinkenden Basenpaarsubstitutionen ver{\"a}ndertes Mutationsspektrum detektiert. Die Auswertung des Mikrokerntests unterst{\"u}tzte die Annahme, dass Irilon Chromosomenmutationenen induziert. Zudem wies Irilon ein starkes aneugenes Potential auf. Im Gegensatz zu den ph{\"a}notypselektiven Mutationstests weisen genotypselektive Tests hingegen theoretisch keine Limitierungen hinsichtlich der zu untersuchenden Zielsequenz und der Organwahl auf. Ein Vertreter der genotypselektiven Tests ist der Random Mutation Capture Assay, der 2005 von Bielas und Loeb f{\"u}r das Intron 6 des humanen TP53-Gens publiziert wurde. Ein weiteres Ziel dieser Arbeit war es zu untersuchen ob die Technik des Random Mutation Capture Assays auf die Ratte {\"u}bertragbar und ob bzw.unter welchen Bedingungen die Bestimmung von spontanen und induzierten Mutationsfrequenzen in verschiedenen Zielsequenzen m{\"o}glich ist. Deshalb wurden zun{\"a}chst das f{\"u}r das Tumorsuppressor Protein 53 kodierenden Gen p53, die f{\"u}r die 18S ribosomale RNA kodierenden DNA und das mitochondriale Cytochrom b Gen als Zielsequenzen gew{\"a}hlt und deren Eignung f{\"u}r die Anwendung im Random Mutation Capture Assays gepr{\"u}ft. F{\"u}r jede Zielsequenz wurden alle f{\"u}r die Durchf{\"u}hrung des Random Mutations Capture Assays ben{\"o}tigten molekularen Werkzeuge unter optimierten PCR-Bedingungen hergestellt und verifiziert. F{\"u}r die Quantifizierung der Gesamtkopiezahl wurde je Zielsequenz eine spezifische Echtzeit-PCR-Methode entwickelt, welche TaqMan®-Sonden-basiert ist. Nach Optimierung der PCR-Bedingungen wurden je Zielsequenz Wiederfindungen im angestrebten Bereich von ca. 90-100\% mit Schwankungen von maximal 20\% erreicht. Ausgenommen hiervon war die f{\"u}r die 18S ribosomale DNA kodierende Zielsequenz. Eine {\"A}nderung der Echtzeit-PCR-Bedingungen f{\"u}hrte zu keiner praktikablen Methode. Daher war diese Zielsequenz, welche trotz geringer DNA-Mengen versprach mehr DNA Kopien zu erhalten und somit die Bestimmung von geringen Mutationsfrequenzen zu erleichtern, nicht im Random Mutation capture Assay anwendbar. F{\"u}r die Wahl einer DNA-Isolierungsmethode wurden 5 Methoden hinsichtlich einer f{\"u}r die Mutationsfrequenz-Bestimmung ausreichenden Kopiezahlausbeute, der Reinheit und des Kosten-/Zeitaufwands verglichen. Mit zwei der f{\"u}nf Methoden wurde aus 100 mg Gewebe die h{\"o}chste nukle{\"a}ren Kopienzahl isoliert, ausreichend um Mutationsfrequenzen im Bereich 1-2*10-7/bp zu bestimmen. Um jedoch die erwarteten Mutationsfrequenzen im Bereich von 1-3*10-8/bp (Intron) bzw. 2-3*10-9/bp (Exon) zu detektieren, w{\"a}ren 2-3 g Gewebe bzw. 3 mg DNA notwendig. Auf Grund der anatomischen Organgewichte w{\"a}re die Durchf{\"u}hrung des nukle{\"a}ren Random Mutation Capture Assays somit auf vereinzelte Organe wie Leber, D{\"u}nndarm und Gehirn beschr{\"a}nkt. Zudem bestanden mit der Hybridisierung und dem Uracil-DNA-Glycosylase-Verdau zwei zus{\"a}tzliche kritische Punkte, welche zu einer Minimierung der Kopiezahl oder einer fehlerhaften Einsch{\"a}tzung der Mutationsfrequenz f{\"u}hren k{\"o}nnen. Aus diesen Gr{\"u}nden wurde eine Entwicklung des Random Mutation Capture Assays f{\"u}r die Zielsequenz im p53-Gen verworfen. Die Kopiezahlausbeuten der mitochondrialen DNA waren ab 50 mg Gewebeeinsatz bei jeder der 5 untersuchten Methoden ausreichend zur Bestimmung einer angestrebten Spontanmutationsfrequenz zwischen 6-100*10-7/bp. Bei Gewebemengen unter 50 mg erwies sich die Aufarbeitung mit DNAzol® auf Grund zu niedriger Kopiezahlausbeuten als ungeeignet. In dieser Arbeit wurde nachfolgend die Phenol-Chloroform-Extraktion nach Vermulst et al (2008) verwendet. Im Rahmen der Etablierung der PCR zur Erfassung der Anzahl mutierter Kopien (Mutations-PCR) wurde ein Mutanten-Standard zur Anwendung als Positivkontrolle in PCR und Agarose-Gelelektrophorese hergestellt, verifiziert und fluorimetrisch quantifiziert. Wiederfindungsexperimente best{\"a}tigten, dass mit der etablierten Mutations-PCR eine einzelne Kopie amplifizier- und detektierbar ist. Um eine Auswertung einer Sequenzierung hinsichtlich Anzahl der Mutanten als auch der Sequenz an sich zu gew{\"a}hrleisten, wurde der akzeptierte Bereich an detektierten 1-19 (80 Reaktionen) gesetzt. Nachfolgend wurde in der gesunden Leber von m{\"a}nnlichen und weiblichen Ratten erfolgreich die mitochondriale Spontanmutationsfrequenz mit dem entwickelten Random Mutation Capture Assay bestimmt. Diese betrug innerhalb einer mitochondrialen DNA-L{\"o}sung 3,2 ± 3,1 *10-6/bp (Median 2,7). Die Mutationsfrequenzen von 3 unabh{\"a}ngigen mitochondrialen DNA-L{\"o}sungen -isoliert aus demselben Organpulver- betrugen durchschnittlich 11,5 ± 8,6 *10-6/bp (Median 8,0) und waren somit ca. 3-mal h{\"o}her. Ein Vergleich zwischen den Mutationsfrequenzen der m{\"a}nnlichen und weiblichen Tiere resultierte in mitochondrialen Mutationsfrequenzen zwischen 1,6-34,4 *10-6/bp (m{\"a}nnlich) und 3,0-12,9 *10-6/bp (weiblich), wobei zwischen m{\"a}nnlichen und weiblichen Tieren kein statistischer Unterschied bestand (Mann-Whitney-Test; p<0,05). Um zu pr{\"u}fen, ob die Mutationsraten bestimmt mit dem mitochondrialen Random Mutation Capture Assay und einem ph{\"a}notypselektiven Mutationstest zu gleichem Maße auf ein mutagenes Potential hinweisen, wurde als n{\"a}chstes der ph{\"a}notypselektive Hypoxanthin-Guanin-Phosphoribosyltransferase-Test f{\"u}r normale Nierenepithelzellen der Ratte (NRK-Zelllinie) entwickelt. Nach einer 24 h Inkubation mit 0,1 µM 4-Nitrochinolin-1-oxid, einem bekannten Adduktbildner, stieg die Mutationsfrequenz im Exon des Hypoxanthin-Guanin-Phosphoribosyltransferase-Gens um den Faktor 5 im Vergleich zur L{\"o}semittelkontrolle an. Mit Hilfe des entwickelten Random Mutation Capture Assays wurde in der DNA -isoliert zum Zeitpunkt der Selektion- eine dreifache Steigerung der Mutationsfrequenz im mt-Cytb-Gen detektiert. Somit war mit beiden Tests eine Erh{\"o}hung der Mutationsfrequenz in der gleichen Gr{\"o}ßenordnung detektierbar, wobei der ph{\"a}notypselektive Mutationstest sensitiver war. Nachdem die Mutations-PCR ca. 1,5 Jahren angewendet wurde, stieg innerhalb von 4 Monaten unabh{\"a}ngig von der verwendeten Templatkonzentration sowohl die H{\"a}ufigkeit der detektierten Schmierbanden als auch die des DNA hang up an. In 7 Mutations-PCRs, welche nach diesen Ph{\"a}nomenen nur mit Blindwerten durchgef{\"u}hrt wurden, lag der Anteil an detektierten DNA-Schmierbanden pro Mutations-PCR zwischen 25,0\% und 38,8\%, der des DNA hang up zwischen 17,5\% und 48,8\%. Das war h{\"a}ufiger als in Reaktionen mit Templat; ein Hinweis daf{\"u}r, dass das Vorliegen von Templat Nebenreaktionen zu einem gewissen Grad verdr{\"a}ngte und dass die unspezifische Amplifizierung am Mastermix der Mutations-PCR lag. Eine {\"A}nderung von chemischen oder physikalischen Parametern innerhalb der PCR-Reaktion f{\"u}hrte zu keiner Reduktion der Nebenprodukte. Somit war der f{\"u}r das mitochondriale Cytochrom b-Gen entwickelte Random Mutation Capture Assay nicht robust gegen{\"u}ber Nebenreaktionen und ist daher nicht f{\"u}r einen routinem{\"a}ßigen Einsatz geeignet. Zusammenfassend war eine Entwicklung der Primer und der molekularen Werkzeuge des Random Mutation Capture Assays vom Mensch auf Ratte mit allen drei gew{\"a}hlten Zielsequenzen m{\"o}glich. Im Rahmen der Experimente zeigte sich, dass die Kopiezahl-PCR der Zielsequenz in der 18S ribosomale RNA kodierenden DNA nicht praktikabel und eine Bestimmung der Mutationsfrequenzen f{\"u}r das Tumorsuppressor Protein 53 kodierenden Gen p53 nur unter Ber{\"u}cksichtigung einer eingeschr{\"a}nkten Organauswahl m{\"o}glich war. F{\"u}r die Zielsequenz des mitochondrialen Cytochrom b Gens war der Random Mutation Capture Assay durchf{\"u}hrbar. Allerdings erwies sich die Mutations-PCR als instabil. Folglich ist eine Bestimmung von Mutationsfrequenzen mit dem Random Mutation Capture Assay in Rattus norvegicus nur sehr begrenzt m{\"o}glich.},
  subject      = {Mutationsrate},
  language  = {de}
}
@phdthesis{Erk2018,
  author    = {Erk, Christine},
  title     = {Metabolismus und Reaktivit{\"a}tsstudien neuer Arzneistoffe mittels LC-MS/MS-Methoden},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-167025},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Diese Arbeit befasst sich mit der Untersuchung des Metabolismus sowie der Reaktivit{\"a}t verschiedener Wirk- und Arzneistoffe mittels fl{\"u}ssigchromatographischer und massen-spektrometrischer Methoden, sie gliedert sich dabei in vier Projekte. Zur Bestimmung des Metabolitenprofils wurde ein passendes In-vitro-Inkubationssystem mit Cytochrom-P-450-Systemen entwickelt. So wurden der Metabolismus und die Pharmakokinetik der Mip-Inhibitoren SF110, SF235 und SF354 gegen Legionellen, sowie neuer antitrypanosomaler Verbindungen MB209, MB343 und MB444 und von Daptomycin bestimmt. Dar{\"u}ber hinaus wurde die antibakterielle Aktivit{\"a}t des Daptomycins gegen{\"u}ber einem unbekannten Staphylokokkus-Stammes S. sciuri ermittelt. Außerdem wurden Reaktivit{\"a}tsuntersuchungen neu synthetisierter Inhibitoren gegen Tuberkulose und S. aureus durchgef{\"u}hrt. Die untersuchten Mip-Inhibitoren lieferten ein Metabolitenprofil, welches durch Ester- und Amidhydrolysen sowie Hydroxylierungen gepr{\"a}gt wurde. Die Verbindung SF110 schien dabei bereits eine gewisse Instabilit{\"a}t der Esterbindung aufzuweisen, da auch im Blindwert entsprechende Spaltprodukte identifiziert werden konnten. Die Hauptmetabolite von SF235 und SF354 bildeten sich durch unterschiedliche Hydrolysen, da die Spaltung des Molek{\"u}ls von den jeweiligen Substituenten abh{\"a}ngig ist. Innerhalb dieser Substanzklasse dominiert die mikrosomale Enzymkatalyse, da der gr{\"o}ßte metabolische Umsatz sowie die meisten Metabolite mittels mikrosomaler Fraktion des Menschen bzw. der Maus gefunden wurden. Die Klasse der Mip-Inhibitoren wird somit vor allem durch Cytochrom-P-450-Enzyme umgesetzt, wobei die Hydrophilie durch Einf{\"u}hrung polarer OH-Gruppen der Molek{\"u}le erh{\"o}ht wird. Die Hydroxylierung scheint dabei positionsspezifisch, bedingt durch sterische Hinderungen oder dirigierende Einfl{\"u}sse, abzulaufen. Stabilit{\"a}tsvergleiche zwischen SF110, SF235 und SF354 zeigten, dass die Einf{\"u}hrung einer Amidbindung anstelle der korrespondierenden Esterbindung die Substanzklasse maßgeblich metabolisch stabilisiert. Im Rahmen des murinen In-vivo-Metabolismus wurde beobachtet, dass SF235 einem deutlich st{\"a}rkeren Metabolismus unterlag als SF354 und sich der Metabolismus vor allem innerhalb der ersten 30 min vollzog. Demgegen{\"u}ber zeigten die In-vitro-Ergebnisse gegenteilige Ergebnisse, bei denen SF354 die am st{\"a}rksten metabolisierte Substanz war. Diese widerspr{\"u}chlichen Ergebnisse deuten darauf hin, dass In-vitro-Modelle nur als Anhaltspunkt verwendet werden sollten, um m{\"o}gliche Trends abzuleiten. Metabolismusstudien der Chinolonamide, die gegen die afrikanische Schlafkrankheit wirken sollen, veranschaulichten, dass die gr{\"o}ßte enzymatische Umsetzung aller drei getesteten Verbindungen mittels cytosolischer Fraktion erfolgte. Die Enzymreaktionen werden vermutlich durch ALDH bzw. MAO dominiert und nicht durch CYP bzw. FMO. Die gebildeten Metabolite in den verschiedenen Fraktionen unterlagen (ω-1)-Oxidationen, N-Desalkylierungen, Amidhydrolysen und aromatischen Hydroxylierungen. Auffallend war, dass eine Hydroxylierung am aromatischen Benzylring nur erfolgen konnte, sofern der Benzylaromat keinen Fluorsubstitutenten trug, da dieser desaktivierend wirkte. Die aromatische Hydroxylierung am Chinolonamid erfolgte dagegen bei allen drei Substanzen. Es wurde somit lediglich eine Hydroxylierung am Benzylring von MB343 festgestellt. Die enzymatische Aktivit{\"a}t aller Substanzen folgte einer Reaktionskinetik 1. Ordnung. Die unterschiedlichen Stabilit{\"a}ten der Substanzen zeigten einen deutlichen Trend: MB209 wurde, da es die instabilste Verbindung darstellt, im gr{\"o}ßten Maße umgesetzt, gefolgt von den stabileren Derivaten MB343 und MB444. Die Untersuchung der enzymatischen Aktivit{\"a}ten zeigte, dass die drei Substanzen, verglichen mit der Leitstruktur GHQ168, eine um den Faktor zehn geringere Aktivit{\"a}t aufwiesen [19]. Aufgrund der eingef{\"u}hrten Fluoratome weisen die Substanzen somit eine wesentlich h{\"o}here Stabilit{\"a}t auf. Diese Ergebnisse wurden durch die Untersuchung der Halbwertszeit best{\"a}tigt, bei der MB444 den h{\"o}chsten Wert besaß. Weiterhin ist die Position des Fluorsubstituenten am Chinolonger{\"u}st ausschlaggebend f{\"u}r die metabolische Stabilit{\"a}t, wobei MB444 aufgrund des para-Fluorsubstituenten am Chinolonamid die stabilste Verbindung darstellt. Durch Inkubation von Daptomycin mit unterschiedlichen S. sciuri-Isolaten wurde ein m{\"o}glicher Inaktivierungsmechanismus beobachtet, bei dem das Antibiotikum durch Spaltung des cyclischen Aminos{\"a}ureringes, durch Deacylierung des Fetts{\"a}ureschwanzes, einer Kombination beider Mechanismen oder durch eine Spaltung des heteroaromatischen Ringsystems von Tryptophan inaktiviert wurde. Die Proteasen des Daptomycin-resistenten S. sciuri-Isolats TS92 f{\"u}hrten zu einem Daptomycinabbau von 35 \%, unabh{\"a}ngig von der eingesetzten Menge des Arzneistoffes. Das Ausmaß des Abbaus scheint dar{\"u}ber hinaus vom eingesetzten Inkubationsmedium abh{\"a}ngig zu sein, da die Proteasen voraussichtlich auf ein bestimmtes N{\"a}hrmedium angewiesen sind. Der sensitive S. sciuri-Stamm TS93 lieferte die h{\"o}chste Abbaurate an Daptomycin mit 55 \% und widerlegt damit die Vermutung, dass Daptomycin die geringste antibakterielle Aktivit{\"a}t gegen{\"u}ber diesem S. sciuri-Stamm aufweist. Im In-vitro-Metabolismus zeigte Daptomycin insgesamt eine sehr geringe Umsetzungsmenge mit maximal 5 \% nach 4 h und einer geringen Metabolitenbildung. Hier wurde nur ein Metabolit gefunden, welcher auch mittels S. sciuri-Inkubation identifiziert wurde. Dieser Mechanismus k{\"o}nnte somit auf anderem Wege verlaufen. Die Reaktivit{\"a}tsstudien der kovalenten Inhibitoren der FadA5-Thiolase gegen Tuberkulose zeigten, dass nur die Verbindungen C1 und C4 eine Reaktivit{\"a}t gegen{\"u}ber der Aminos{\"a}ure Cystein93 im aktiven Zentrum besaßen, die somit f{\"u}r den gew{\"u}nschten Einsatzzweck geeignet sein k{\"o}nnten. Weiterhin wurde bei den kovalenten Inhibitoren der Enoyl-ACP-Reduktase mit dem Enzym FabI, welches im aktiven Zentrum ein Tyrosin besitzt, keine Reaktion festgestellt, da keine Addukte identifiziert wurden. Dies ist vermutlich auf die Unl{\"o}slichkeit im verwendeten TRIS-Puffer zur{\"u}ckzuf{\"u}hren.},
  subject      = {Biotransformation},
  language  = {de}
}
@phdthesis{Gador2018,
  author    = {Gador, Eva},
  title     = {Strategies to improve the biological performance of protein therapeutics},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161798},
  school      = {Universit{\"a}t W{\"u}rzburg},
  pages     = {199},
  year      = {2018},
  abstract  = {During the last decades the number of biologics increased dramatically and several biopharmaceutical drugs such as peptides, therapeutic proteins, hormones, enzymes, vaccines, monoclonal antibodies and antibody-drug conjugates conquered the market. Moreover, administration and local delivery of growth factors has gained substantial importance in the field of tissue engineering. Despite progress that has been made over the last decades formulation and delivery of therapeutic proteins is still a challenge. Thus, we worked on formulation and delivery strategies of therapeutic proteins to improve their biological performance. Phase I of this work deals with protein stability with the main focus on a liquid protein formulation of the dimeric fusion protein PR-15, a lesion specific platelet adhesion inhibitor. In order to develop an adequate formulation ensuring the stability and bioactivity of PR-15 during storage at 4 °C, a pH screening, a forced degradation and a Design of Experiments (DoE) was performed. First the stability and bioactivity of PR-15 in 50 mM histidine buffer in relation to pH was evaluated in a short-term storage stability study at 25 °C and 40 °C for 4 and 8 weeks using different analytical methods. Additionally, potential degradation pathways of PR-15 were investigated under stressed conditions such as heat treatment, acidic or basic pH, freeze-thaw cycles, light exposure, induced oxidation and induced deamidation during the forced degradation study. Moreover, we were able to identify the main degradation product of PR-15 by performing LC/ESI-MS analysis. Further optimization of the injectable PR 15 formulation concerning pH, the choice of buffer and the addition of excipients was studied in the following DoE and finally an optimal PR-15 formulation was found. The growth factors BMP-2, IGF-I and TGF-β3 were selected for the differentiation of stem cells for tissue engineering of cartilage and bone in order to prepare multifunctionalized osteochondral implants for the regeneration of cartilage defects. Silk fibroin (SF) was chosen as biomaterial because of its biocompatibility, mechanical properties and its opportunity for biofunctionalization. Ideal geometry of SF scaffolds with optimal porosity was found in order to generate both tissues on one scaffold. The growth factors BMP-2 and IGF-I were modified to allow spatially restricted covalent immobilization on the generated porous SF scaffolds. In order to perform site-directed covalent coupling by the usage of click chemistry on two opposite sides of the scaffold, we genetically engineered BMP-2 (not shown in this work; performed by Barbara Tabisz) and IGF-I for the introduction of alkyne or azide bearing artificial amino acids. TGF β3 was immobilized to beads through common EDC/NHS chemistry requiring no modification and distributed in the pores of the entire scaffold. For this reason protein modification, protein engineering, protein immobilization and bioconjugation are investigated in phase II. Beside the synthesis the focus was on the characterization of such modified proteins and its conjugates. The field of protein engineering offers a wide range of possibilities to modify existing proteins or to design new proteins with prolonged serum half-life, increased conformational stability or improved release rates according to their clinical use. Site-directed click chemistry and non-site-directed EDC/NHS chemistry were used for bioconjugation and protein immobilization with the aim to underline the preferences of site-directed coupling. We chose three strategies for the incorporation of alkyne or azide functionality for the performance of click reaction into the protein of interest: diazonium coupling reaction, PEGylation and genetic engineering. Azido groups were successfully introduced into SF by implementation of diazonium coupling and alkyne, amino or acid functionality was incorporated into FGF-2 as model protein by means of thiol PEGylation. The proper folding of FGF-2 after PEGylation was assessed by fluorescence spectroscopy, WST-1 proliferation assay ensured moderate bioactivity and the purity of PEGylated FGF-2 samples was monitored with RP-HPLC. Moreover, the modification of native FGF-2 with 10 kDa PEG chains resulted in enhanced thermal stability. Additionally, we genetically engineered one IGF-I mutant by incorporating the unnatural amino acid propargyl-L-lysine (plk) at position 65 into the IGF-I amino acid sequence and were able to express hardly verifiable amounts of plk-IGF-I. Consequently, plk-IGF-I expression has to be further optimized in future studies in order to generate plk-IGF-I with higher yields. Bioconjugation of PEGylated FGF-2 with functionalized silk was performed in solution and was successful for click as well as EDC/NHS chemistry. However, substantial amounts of unreacted PEG-FGF-2 were adsorbed to SF and could not be removed from the reaction mixture making it impossible to expose the advantages of click chemistry in relation to EDC/NHS chemistry. The immobilization of PEG-FGF-2 to microspheres was a trial to increase product yield and to remove unreacted PEG-FGF-2 from reaction mixture. Bound PEG-FGF-2 was visualized by fluorescence imaging or flow cytometry and bioactivity was assessed by analysis of the proliferation of NIH 3T3 cells. However, immobilization on beads raised the same issue as in solution: adsorption caused by electrostatic interactions of positively charged FGF-2 and negatively charged SF or beads. Finally, we were not able to prove superiority of site-directed click chemistry over non-site-directed EDC/NHS. The skills and knowledge in protein immobilization as well as protein characterization acquired during phase II helped us in phase III to engineer cartilage tissue in biofunctionalized SF scaffolds. The approach of covalent immobilization of the required growth factors is relevant because of their short in vivo half-lives and aimed at controlling their bioavailability. So TGF-β3 was covalently coupled by means of EDC/NHS chemistry to biocompatible and biostable PMMA beads. Herein, we directly compared bioactivity of covalently coupled and adsorbed TGF-β3. During the so-called luciferase assay bioactivity of covalent coupled as well as adsorbed TGF-β3 on PMMA beads was ensured. In order to investigate the real influence of EDC/NHS chemistry on TGF-β3's bioactivity, the amount of immobilized TGF-β3 on PMMA beads was determined. Therefore, an ELISA method was established. The assessment of total amount of TGF-β3 immobilized on the PMMA beads allowed as to calculate coupling efficiency. A significantly higher coupling efficiency was determined for the coupling of TGF-β3 via EDC/NHS chemistry compared to the reaction without coupling reagents indicating a small amount of adsorbed TGF-β3. These results provide opportunity to determine the consequence of coupling by means of EDC/NHS chemistry for TGF β3 bioactivity. At first sight, no statistically significant difference between covalent immobilized and adsorbed TGF-β3 was observed regarding relative luciferase activities. But during comparison of total and active amount of TGF-β3 on PMMA beads detected by ELISA or luciferase assay, respectively, a decrease of TGF-β3's bioactivity became apparent. Nevertheless, immobilized TGF β3 was further investigated in combination with SF scaffolds in order to drive BMSCs to the chondrogenic lineage. According to the results obtained through histological and immunohistochemical studies, biochemical assays as well as qRT-PCR of gene expression from BMSCs after 21 days in culture immobilized TGF-β3 was able to engineer cartilage tissue. These findings support the thesis that local presentation of TGF β3 is superior towards exogenous TGF β3 for the development of hyaline cartilage. Furthermore, we conclude that covalent immobilized TGF β3 is not only superior towards exogenously supplemented TGF-β3 but also superior towards adsorbed TGF-β3 for articular hyaline cartilage tissue engineering. Diffusion processes were inhibited through covalent immobilization of TGF-β3 to PMMA beads and thereby a stable and consistent TGF-β3 concentration was maintained in the target area. With the knowledge acquired during phase II and III as well as during the studies of Barbara Tabisz concerning the expression and purification of plk-BMP-2 we made considerable progress towards the formation of multifunctionalized osteochondral implants for the regeneration of cartilage defects. However, further studies are required for the translation of these insights into the development of multifunctionalized osteochondral SF scaffolds.},
  subject      = {biologics},
  language  = {en}
}
@phdthesis{Braun2018,
  author    = {Braun, Alexandra Carolin},
  title     = {Bioresponsive delivery of anticatabolic and anabolic agents for muscle regeneration using bioinspired strategies},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-169047},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Progressive loss of skeletal muscle mass, strength and function poses a major threat to independence and quality of life, particularly in the elderly. To date, sarcopenia therapy consists of resistance exercise training in combination with protein supplementation due to the limited efficacy of available pharmacological options in counteracting the effects of muscle wasting. Therapeutic intervention with growth factors including insulin-like growth factor I (IGF-I) or inhibitors of myostatin  a potent suppressor of myogenesis  hold potential to rebalance the altered activity of anabolic and catabolic cytokines. However, dosing limitations due to acute side effects and disruptions of the homeostasis have so far precluded clinical application. Intending to provide a therapy with a superior safety and efficacy profile by directing drug release to inflamed tissue and minimizing off-target activity, we designed bioresponsive delivery systems for an anti-catabolic peptide and anabolic IGF-I responding to local flares of muscle wasting. In Chapter I, current concepts for bioorthogonal conjugation methods are discussed and evaluated based on various drug delivery applications. With a focus on protein delivery, challenges and potential pitfalls of each chemical and enzymatic conjugation strategy are analyzed and opportunities regarding their use for coupling of biomolecules are given. Based on various studies conjugating proteins to polymers, particles and biomaterials using different site-directed approaches, the chapter summarizes available strategies and highlights certain aspects requiring particular consideration when applied to biomolecules. Finally, a decision process for selection of an optimum conjugation strategy is exemplarily presented. Three of these bioorthogonal coupling reactions are applied in Chapter II detailing the potential of site-directed conjugation in the development of novel, homogenous drug delivery systems. The chapter describes the design of a delivery system of a myostatin inhibitor (MI) for controlled and local release counteracting myositis flares. MI release from the carrier is driven by increased matrix metalloproteinase (MMP) levels in compromised muscle tissues cleaving the interposed linker, thereby releasing the peptide inhibitor from the particulate carrier. Release experiments were performed to assess the response towards various MMP isoforms (MMP-1, -8, -9 and -13) - as upregulated during skeletal muscle myopathies - and the release pattern of the MI in case of disease progression was analyzed. By selection of the protease-sensitive linker (PSL) showing variable susceptibilities to proteases, release rates of the MI can be controlled and adapted. Immobilized MI as well as released MI as response to MMP upregulation was able to antagonize the effects of myostatin on cell signalling and myoblast differentiation. The approach of designing bioresponsive protein delivery systems was also applied to the anabolic growth factor IGF-I, as described in Chapter III. Numerous studies of PEGylated proteins or peptides reveal, that successful therapy is challenged by safety and efficacy issues, as polymer attachment considerably alters the properties of the biologic, thereby jeopardizing clinical efficacy. To this end, a novel promising approach is presented, intending to exploit beneficial effects of PEGylation on pharmacokinetics, but addressing the pharmacodynamic challenges by releasing the protein upon entering the target tissue. This was realized by integration of a PSL between the PEG moiety and the protein. The soluble polymer conjugate was produced by site-directed, enzymatic conjugation of IGF-I to the PSL, followed by attachment of a 30 kDa-PEG using Strain-promoted azide-alkyne cycloaddition (SPAAC). This strategy illustrates the potential of bioorthogonal conjugation (as described in Chapter I) for generation of homogenous protein-polymer conjugates with reproducible outcome, but also emphasizes the altered protein properties resulting from permanent polymer conjugation. As compared to wild type IGF-I, the PEGylated protein showed considerable changes in pharmacologic effects - such as impaired insulin-like growth factor binding protein (IGFBPs) interactions, submaximal proliferative activity and altered endocytosis patterns. In contrast, IGF-I characteristics were fully restored upon local disintegration of the conjugate triggered by MMP upregulation and release of the natural growth factor. For successful formulation development for the proteins and conjugates, the careful selection of suitable excipients is crucial for a safe and reliable therapy. Chapter IV addresses one aspect by highlighting the chemical heterogeneity of excipients and associated differences in performance. Polysorbate 80 (PS80) is a surfactant frequently used in protein formulations to prevent aggregation and surface adsorption. Despite being widely deployed as a standard excipient, heterogeneous composition and performance entails the risk of eliciting degradation and adverse effects on protein stability. Based on a comprehensive study using different batches of various suppliers, the PS80 products were characterized regarding chemical composition and physicochemical properties, facilitating the assessment of excipient performance in a formulation. Noticeable deviations were recorded between different suppliers as well as between batches of the same suppliers. Correlation of all parameters revealed, that functionality related characteristics (FRCs) could be reliably predicted based on chemical composition alone or by a combination of chemical and physicochemical properties, respectively. In summary, this thesis describes and evaluates novel strategies for the targeted delivery and controlled release of biologics intended to counteract the imbalance of anabolic and catabolic proteins observed during aging and musculoskeletal diseases. Two delivery platforms were developed and characterized in vitro - (i) using anti-catabolic peptides immobilized on a carrier for local delivery and (ii) using soluble IGF-I polymer conjugates for systemic application. Both approaches were implemented by bioorthogonal coupling strategies, which were carefully selected in consideration of limitations, side reactions and efficiency aspects. Bioresponsive release of the active biomolecules following increased protease activity could be successfully realized. The therapeutic potential of these approaches was demonstrated using various cell-based potency assays. The systems allow targeted and controlled release of the growth factor IGF-I and anti-catabolic peptides thereby overcoming safety concerns of current growth factor therapy and thus positively impacting the benefit-risk profile of potent therapeutics. Taking potential heterogeneity and by-product concerns into account, comprehensive excipient characterization was performed and a predictive algorithm for FRCs developed, in order to facilitate formulation design and guarantee a safe and efficient therapy from start to finish.},
  subject      = {Muskelatrophie},
  language  = {en}
}
@phdthesis{Schramm2018,
  author    = {Schramm, Simon},
  title     = {Synthesis of Dualsteric Muscarinic M\(_1\) Acetylcholine Receptor Ligands and Neuroprotective Esters of Silibinin},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173592},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Alzheimer's disease is a complex network of several pathological hallmarks. These characteristics always occur concomitantly and cannot be taken as distinct features of the disease. While there are hypotheses trying to explain the origin and progression of the illness, none of them is able to pinpoint a definitive cause. This fact challenges researchers not to focus on one individual hallmark but, bearing in mind the big picture, target two or more indications at once. This work, therefore, addresses two of the major characteristics of AD: the cholinergic hypothesis and neurotoxic oxidative stress. The former was achieved by targeting the postsynaptic muscarinic M1 acetylcholine receptor to further investigate its pharmacology, and the latter with the synthesis of neuroprotective natural antioxidant hybrids. The first aim was the design and synthesis of dualsteric agonists of the muscarinic M1 acetylcholine receptor. Activation of this receptor was previously shown to improve AD pathologies like the formation of Aβ and NFTs and protect against oxidative stress and caspase activation. Selectively targeting the M1 receptor is difficult as subtypes M1 - M5 of the muscarinic AChRs largely share the same orthosteric binding pocket. Orthosteric ligands are thus unsuitable for selective activation of one specific subtype. Secondary, allosteric binding sites are more diverse between subtypes. Allosteric ligands are, however, in most cases dependent on an orthosteric ligand to cause downstream signals. Dualsteric ligands thus utilize the characteristics of both orthosteric and allosteric ligands in form of a message-address concept. Bridged by an alkylene-linker, the allosteric part ensures selectivity, whereas the orthosteric moiety initiates receptor activation. Two sets of compounds were synthesised in this sense. In both cases, the orthosteric ligand carbachol is connected to an allosteric ligand via linkers of different chain length. The first set utilizes the selective allosteric M1 agonist TBPB, the second set employs the selective M1 positive allosteric modulator BQCA. Six compounds were obtained in twelve-step syntheses each. For each one, a reference compound lacking the carbachol moiety was synthesised. The dualsteric ligands 1a-c and 2a c were tested in the IP1 assay. The assay revealed that the TBPB-dualsterics 1 are not able to activate the receptor, whereas the respective TBPB-alkyl reference compounds 27 gave signals depending on the length of the alkylene-linker, suggesting allosteric partial agonism of alkyl compounds 27 and no dualsteric binding of the putatively dualsteric compounds 1. The dualsteric BQCA molecules 2, however, activated the receptor as expected. Efficacy of the C5 linked compound 2b was the highest, yet C3 and C8 compounds (2a and 2c) also showed partial agonism. In this case, the reference compounds 31 showed no receptor activation, implying the intended dualsteric binding mode of the BQCA-carbachol compounds 2. Further investigations will be conducted by the working group of Dr. Christian Tr{\"a}nkle at the Department of Pharmacology at the University of Bonn to confirm binding modes and determine affinities as well as selectivity of the synthesised dualsteric compounds. The second project dealt with the design, synthesis and biological evaluation of neuroprotective esters of the flavonolignan silibinin. While silibinin is already a potent antioxidant, it has been observed that the 7-OH group has a pro-oxidative character, making this position attractive for functionalisation. In order to obtain more potent antioxidants, the pro-oxidative position was esterified with other antioxidant moieties like ferulic acid 35 and derivatives thereof. Seventeen esters of silibinin 32, including pure diastereomers of 7 O feruloylsilibinin (43a and 43b) and a cinnamic acid ester of 2,3-dehydrosilibinin 46, were synthesised by regioselective esterification using acyl chlorides under basic conditions. The physicochemical antioxidant properties were assessed in the FRAP assay. This assay revealed no improvement of the antioxidant properties except for 7-O-dihydrosinapinoylsilibinin 39b. These results, however, do not correlate with the neuroprotective properties determined in the HT-22 hippocampal neuronal cell model. The assay showed overadditive neuroprotective effects of the esters exceeding those of its components and equimolar mixtures with the most potent compounds being 7-O-cinnamoylsilibinin 37a, 7-O-feruloylsilibinin 38a and the acetonide-protected caffeic acid ester 40a. These potent Michael system bearing compounds may be considered as "PAINS", but the assays used to assess antioxidant and neuroprotective activities were carefully chosen to avoid false positive readouts. The most potent compounds 37a and 38a, as well as the diastereomers 43a and 43b, were further studied in assays related to AD. In vitro ischemia, inhibition of microglial activation, PC12 cell differentiation and inhibition of Aβ42 and τ protein aggregation assays showed similar results in terms of overadditive effects of the synthesised esters. Moreover, the diastereomers 43a and 43b showed differences in their activities against oxytosis (glutamate-induced apoptosis), inhibition of Aβ42 and τ protein aggregation, and PC12 cell differentiation. The stereospecific effect or mode of action against Aβ42 and τ protein aggregation is more pronounced than that of silybin A (32a) and silybin B (32b) reported in literature and needs to be elucidated in future work. Stability measurements in cell culture medium revealed that the esters do not only get hydrolysed but are partially oxidised to their respective 2,3-dehydrosilibinin esters. Because dehydrosilibinin 45 itself is described as a more potent antioxidant than silibinin 32, 7 O cinnamoyl-2,3-dehydrosilibinin 46 was expected to be even more potent than its un-oxidised counterpart 37a in terms of neuroprotection. The oxytosis assay, however, showed that the neurotoxicity of 46 is much more pronounced, especially at higher concentrations, reducing its neuroprotective potential. Dehydrosilibinin esters are therefore inferior to the silibinin esters for application as neuroprotectants, because of the difficulty of their synthesis and their increased neurotoxicity. A synergistic effect of both species (silibinin and the oxidised form) might, however, be possible or even necessary for the pronounced neuroprotective effects of silibinin esters. As the dehydro-species show distinct neuroprotective properties at low concentrations, their continuous formation over time might make an essential contribution to the overall neuroprotection of the synthesised esters. Due to solubility issues for some of the ester compounds, 7-O-cinnamoylsilibinin 37a was converted into a highly soluble hemisuccinate. The vastly improved solubility of 7 O cinnamoyl-23-O-succinylsilibinin 48 was confirmed in shake-flask experiments. Contrary to expectation, stability examinations showed that the succinyl compound 48 is not cleaved to form 7-O-cinnamoylsilibinin 37a. Neuroprotection assays confirmed that 48 is not a prodrug of the corresponding ester. It was determined that the main site of hydrolysis is the 7-position, cleaving 37 to silibinin 32 and cinnamic acid thus reducing the compound's neuroprotective effects. Nevertheless, the compound still showed neuroprotection at a concentration of 25 µM. The improved solubility might be more beneficial than the higher neuroprotection of the poorly soluble parent compound 37a in vivo. 7 O Cinnamoylsilibinin 37a was further investigated to reduce Aβ25 35 induced learning impairment in mice. While tendencies of improved short-term and long-term memory in the animals were observed, the effects are not yet statistically significant in both Y-maze and passive avoidance tests. A greater number of test subjects is necessary to ensure correctness of the preliminary results presented in this work. However, an effect of ester 37a is observable in vivo, showing blood-brain barrier penetration. The esters synthesised are a novel approach for the treatment of AD as they show strong neuroprotective effects and their hydrolysis products or metabolites are only non-toxic natural products.},
  subject      = {Organische Synthese},
  language  = {en}
}
@phdthesis{Hohner2018,
  author    = {Hohner, Matthias Markus},
  title     = {Risikostratifizierung kardialer Nebenwirkungen in der Psychopharmakotherapie \& Entwicklung und Validierung der Dried-Blood-Spot-Analytik f{\"u}r Clozapin und Quetiapin},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-169054},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {1 Verl{\"a}ngerung der kardialen Repolarisationsdauer unter psychiatrischer Medikation bei gleichzeitigem genetischen Basisrisiko Vielen Psychopharmaka wird eine repolarisationsverl{\"a}ngernde Wirkung zugeschrieben. Diese unerw{\"u}nschte Arzneimittelwirkung, erkennbar an einer Verl{\"a}ngerung des QT-Intervalls im Elektrokardiogramm, ist in den vergangenen Jahren, aufgrund des Zusammenhanges mit lebensbedrohlichen Torsades-de-Pointes-Tachyarrhythmien, in den Fokus der klinischen Forschung ger{\"u}ckt. Aufgrund dieser Nebenwirkung werden viele gut wirksame Arzneimittel einer erneuten eingehenden Nutzen-Risiko-Analyse unterzogen und in manchen F{\"a}llen f{\"u}hrte dies zu einer Limitierung der pharmakologischen M{\"o}glichkeiten. Als Hauptmechanismus f{\"u}r eine Psychopharmaka-induzierte QT-Zeit-Verl{\"a}ngerung gilt die Blockade von kardialen Kaliumkan{\"a}len. Aber auch genetische Ver{\"a}nderungen unterschiedlicher kardialer Ionenkan{\"a}le gelten als Risikofaktoren, ebenso wie Effekte anderer ionenabh{\"a}ngiger Signalwege. Da Patienten mit genetischer Pr{\"a}disposition ein defacto erh{\"o}htes Risiko f{\"u}r eine pharmakologisch induzierte QT-Zeit-Verl{\"a}ngerung aufweisen, spricht man von reduzierter Repolarisationsreserve, mit erh{\"o}htem Basislinienrisiko f{\"u}r kardiale Nebenwirkungen. Ziel war es, {\"u}ber einen additiven genetischen Risikoscore eine Quantifizierung individueller Vulnerabilit{\"a}t zu erreichen und zu zeigen, dass dieses Risiko durch die Kontrolle von Medikamenten-Serumspiegeln modulierbar sein kann. Aus einer prospektiven Studie, mit 2062 an endogener Psychose leidenden Patienten des Zentrums f{\"u}r Psychische Gesundheit des Universit{\"a}tsklinikums W{\"u}rzburg, wurden 392 Patienten (mittleres Alter bei Studieneinschluss 41,0 ± 15,0 Jahre, 36,2 \% Frauen) rekrutiert. Prim{\"a}res Einschlusskriterium f{\"u}r die angekn{\"u}pfte, retrospektive Studie war das Vorliegen einer Serumspiegelbestimmung der psychiatrischen Medikation binnen drei Tagen vor oder nach einer elektrokardiographischen Untersuchung (N = 392). Die den Einschlusskriterien entsprechenden 392 Patienten wurden daraufhin auf 62 Einzelpolymorphismen, die in Verbindung mit einer verl{\"a}ngerten QT-Zeit stehen, getestet und die Ergebnisse mit den patientenspezifischen Daten aus den elektrokardiographischen Untersuchungen korreliert. Des Weiteren wurden, basierend auf vier großen Publikationen des internationalen „Cardiac Safety Consortium" (77-79, 148), bekannte polygene Risikoscores, die diese Risikopolymorphismen enthalten, anhand des eigenen Patientenkollektivs berechnet und durch Korrelation mit der QT-Zeit {\"u}berpr{\"u}ft. Diese Scores funktionieren jeweils nach einem Additionsmodell, bei dem nach unterschiedlicher Gewichtung das individuelle Risiko, das durch das Vorhandensein eines bekannten Risikopolymorphismus quantifizierbar wird, zu einem Gesamtrisiko aufsummiert wird. Dar{\"u}ber hinaus ist das Patientenkollektiv auf einen Zusammenhang zwischen dem Serumspiegel der psychiatrischen Medikation und der QT-Zeit gepr{\"u}ft worden. Dazu wurde das Gesamtkollektiv in medikamentenspezifische Subgruppen unterteilt (Amitriptylin (N = 106), Clomipramin (N = 48), Doxepin (N = 53), Mirtazapin (N = 45), Venlafaxin (N = 50), Aripiprazol (N = 56), Clozapin (N = 127), Haloperidol (N = 41), Olanzapin (N = 37), Perazin (N = 47), Quetiapin (N = 119) und Risperidon (N = 106)). Abschließend wurden die Subkollektive in einem kombinierten Rechenmodell daraufhin gepr{\"u}ft, ob Zusammenh{\"a}nge zwischen den genetischen Risikoscores nach Strauss et al. (148) mit dem jeweiligen Medikamenten-Serumspiegel auf die QT-Zeit bestehen. 13 der 62 untersuchten Einzelpolymorphismen zeigten einen signifikanten Zusammenhang mit einer verl{\"a}ngerten Repolarisationsdauer. Ebenfalls korrelieren polygene Risikoscores einer verl{\"a}ngerten kardialen Repolarisation und erkl{\"a}ren einen dabei signifikanten Anteil der Varianz. Die Ergebnisse der Literatur, bez{\"u}glich der Scores nach Pfeufer et al. (77) (R = 0,124, p = 0,014; N = 392), nach Noseworthy et al. (79) (R = 0,169; p = 0,001; N = 392), sowie nach Strauss et al. (148) (R = 0,199; p = 0,000; N = 392) konnten anhand des eigenen Kollektives reproduziert werden, wohingegen der Score von Newton-Cheh et al. (78) keinen signifikanten Zusammenhang mit der QT-Zeit zeigte (R = 0,029; p = 0,568; N = 392). In der Subgruppenanalyse konnte ein stark vom Serumspiegel abh{\"a}ngiger, verl{\"a}ngernder Effekt auf die QT-Zeit f{\"u}r die Arzneistoffe Amitriptylin, Nortriptylin, Clomipramin, und Haloperidol nachgewiesen werden. Die Analyse der mit Amitriptylin behandelten Patienten (N = 106) ergab f{\"u}r Nortriptylin (F (1,104) = 5.986; p = .016, R = .233), als auch f{\"u}r den Summenspiegel aus Amitriptylin und Nortriptylin (F (1,104) = 4.408, p = .038, R = .202) einen signifikanten, nach Cohen einen mittelstarken Zusammenhang mit der QT-Zeit. Starke Effekte auf die QT-Zeit wurden im Zusammenhang mit den Serumspiegeln der Medikamente Clomipramin (F (1,46) = 39.589, p < .001, R = .680, N = 48) und Haloperidol (F (1,39) = 12.672, p = .001, korrigiertes R2= .245, N = 41) errechnet. Ein kombiniertes Rechenmodell, das sowohl den Einfluss des jeweiligen Serumspiegels, als auch des genetischen Risikoscores nach Strauss et al. (148) ber{\"u}cksichtigte, erlaubte bei diesen Arzneistoffen eine signifikant h{\"o}here Varianzaufkl{\"a}rung der QT-Zeit, als die jeweiligen Effekte f{\"u}r sich genommen. Die QT-Zeit gilt als erwiesenermaßen genauso abh{\"a}ngig von der individuellen genetischen Ausstattung, wie auch von Serumspiegeln potentiell als QT-verl{\"a}ngernd eingestufter Medikamente. Diese Effekte scheinen additiv verkn{\"u}pfbar, so dass das von Roden et al. entwickelte Konzept der reduzierten Repolarisationsreserve (54) als best{\"a}tigt gelten darf. Die jeweiligen Einzeleffekte vom genetischen Risiko, sowie der Medikation haben zusammen einen gr{\"o}ßeren Einfluss auf die gemessenen QT-Zeit als f{\"u}r sich alleine genommen. Durch die Genetik l{\"a}sst sich somit tats{\"a}chlich eine grobe vorab-Risikoabsch{\"a}tzung treffen. Dies k{\"o}nnte nach sorgf{\"a}ltiger Nutzen-Risiko-Analyse durch Kontrollen des EKGs und des Serumspiegels moduliert werden und somit vielf{\"a}ltigere therapeutische M{\"o}glichkeiten erhalten. 2 Entwicklung und Validierung einer Dried-Blood-Spot-Methode zum therapeutischen Drug Monitoring von Clozapin und Quetiapin Die Technik der Extraktion und Analyse von Stoffen aus getrocknetem Blut ist bereits seit den 1960er Jahren bekannt, wurde bis zur j{\"u}ngeren Vergangenheit aber eher zu diagnostischen Zwecken angewendet. Durch Fortschritte in der Analytik im Sinne ausgefeilterer Chromatographie und sensitiverer Detektion wurde das Verfahren der Dried-Blood-Spot-Analytik auch f{\"u}r die Spiegelbestimmung von Arzneistoffen interessant. So wurden auch im Bereich des Therapeutischen Drug Monitorings bereits Methoden, beispielsweise f{\"u}r Antibiotika, Antiepileptika, Virostatika und in j{\"u}ngerer Zeit auch Antidiabetika publiziert. Die Vorteile in der Probenhandhabung und durch geringeren Aufwand bei der Blutentnahme sowie geringeres Probenentnahmevolumen werden durch weitere Fortschritte im Bereich der Analytik vordergr{\"u}ndiger. Ziel war es, ein Extraktionsverfahren zu entwickeln und zu validieren, dass die gemeinsame Quantifizierung der h{\"a}ufig verabreichten Antipsychotika Clozapin und Quetiapin aus einem einzelnen getrockneten Blutstropfen erm{\"o}glicht. Die Extraktion mit einer Mischung aus 99 \% Acetonitril und 1 \% 1 M Salzs{\"a}ure und anschließender HPLC-Analyse mit S{\"a}ulenschaltung und photometrischer Detektion wurde nach den Richtlinien der Gesellschaft f{\"u}r toxikologische und forensische Chemie (GTFCh) (146) validiert. Sie entsprach s{\"a}mtlichen Anforderungen bez{\"u}glich Linearit{\"a}t, Bestimmungsgrenze, Stabilit{\"a}t, Genauigkeit, Extraktionsausbeute und Robustheit. Somit gilt diese Methode in der Praxis als anwendbar und d{\"u}rfte, nach {\"U}berpr{\"u}fung der therapeutischen Bereiche f{\"u}r kapillares Vollblut im Vergleich zu den bereits definierten Bereichen f{\"u}r ven{\"o}se entnommene Serumproben, Eingang in die klinische Praxis finden.},
  subject      = {Pharmakotherapie},
  language  = {de}
}