@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{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{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}
}
@article{ChenHiranoWerneretal.2018,
  author    = {Chen, Xinyu and Hirano, Mitsuru and Werner, Rudolf A. and Decker, Michael and Higuchi, Takahiro},
  title     = {Novel \(^{18}\)F-labeled PET Imaging Agent FV45 targeting the Renin-Angiotensin System},
  series = {ACS Omega},
  volume    = {3},
  journal   = {ACS Omega},
  number    = {9},
  issn      = {2470-1343},
  doi       = {10.1021/acsomega.8b01885},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-167144},
  pages     = {10460-10470},
  year      = {2018},
  abstract  = {Renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure and hormonal balance. Using positron emission tomography (PET) technology, it is possible to monitor the physiological and pathological distribution of angiotensin II type 1 receptors (AT\(_1\)), which reflects the functionality of RAS. A new \(^{18}\)F-labeled PET tracer derived from the clinically used AT\(_1\) antagonist valsartan showing the least possible chemical alteration from the valsartan structure has been designed and synthesized with several strategies, which can be applied for the syntheses of further derivatives. Radioligand binding study showed that the cold reference FV45 (K\(_i\) 14.6 nM) has almost equivalent binding affinity as its lead valsartan (K\(_i\) 11.8 nM) and angiotensin II (K\(_i\) 1.7 nM). Successful radiolabeling of FV45 in a one-pot radiofluorination followed by the deprotection procedure with 21.8 ± 8.5\% radiochemical yield and >99\% radiochemical purity (n = 5) enabled a distribution study in rats and opened a path to straightforward large-scale production. A fast and clear kidney uptake could be observed, and this renal uptake could be selectively blocked by pretreatment with AT\(_1\)-selective antagonist valsartan. Overall, as the first \(^{18}\)F-labeled PET tracer based on a derivation from clinically used drug valsartan with almost identical chemical structure, [\(^{18}\)F]FV45 will be a new tool for assessing the RAS function by visualizing AT\(_i\) receptor distributions and providing further information regarding cardiovascular system malfunction as well as possible applications in inflammation research and cancer diagnosis.},
  subject      = {Positronen-Emissions-Tomografie},
  language  = {en}
}
@article{RasheedHoelleinHolzgrabe2018,
  author    = {Rasheed, Huma and H{\"o}llein, Ludwig and Holzgrabe, Ulrike},
  title     = {Future information technology tools for fighting substandard and falsified medicines in low- and middle-income countries},
  series = {Frontiers in Pharmacology},
  volume    = {9},
  journal   = {Frontiers in Pharmacology},
  number    = {995},
  doi       = {10.3389/fphar.2018.00995},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-177068},
  year      = {2018},
  abstract  = {Substandard and falsified (SF) medicines have emerged as a global public health issue within the last two decades especially in low- and middle-income countries (LMICs). Serious consequences of this problem include a loss of trust and increased financial costs due to less disease control and more frequent complications during therapy. Of note, antimicrobial resistance is an additional long-term implication of poor-quality antimicrobials. This review covers information technology tools including medicines authentication tools (MAT) as mobile apps and messaging service, 2D barcoding approaches with drug safety alert systems, web based drug safety alerts, radiofrequency identification tags, databases to support visual inspection, digital aids to enhance the performance of quality evaluation kits, reference libraries for identification of falsified and substandard medicines, and quality evaluation kits based on machine learning for field testing. While being easy to access and simple to use, these initiatives are gaining acceptance in LMICs. Implementing 2D barcoding based on end-to-end verification and "Track and Trace" systems has emerged as a step toward global security in the supply chain. A breakthrough in web-based drug safety alert systems and data bases was the establishment of the Global Surveillance and Monitoring System by the World Health Organization in 2013. Future applications include concepts including "lab on a chip" and "paper analytical devices" and are claimed to be convenient and simple to use as well as affordable. The principles discussed herein are making profound impact in the fight against substandard and falsified medicines, offering cheap and accessible solutions.},
  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{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}
}
@article{VolpatoHolzgrabe2018,
  author    = {Volpato, Daniela and Holzgrabe, Ulrike},
  title     = {Designing Hybrids Targeting the Cholinergic System by Modulating the Muscarinic and Nicotinic Receptors: A Concept to Treat Alzheimer's Disease},
  series = {Molecules},
  volume    = {23},
  journal   = {Molecules},
  number    = {12},
  issn      = {1420-3049},
  doi       = {10.3390/molecules23123230},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-197555},
  pages     = {3230},
  year      = {2018},
  abstract  = {The cholinergic hypothesis has been reported first being the cause of memory dysfunction in the Alzheimer's disease. Researchers around the globe have focused their attention on understanding the mechanisms of how this complicated system contributes to processes such as learning, memory, disorientation, linguistic problems, and behavioral issues in the indicated chronic neurodegenerative disease. The present review reports recent updates in hybrid molecule design as a strategy for selectively addressing multiple target proteins involved in Alzheimer's disease (AD) and the study of their therapeutic relevance. The rationale and the design of the bifunctional compounds will be discussed in order to understand their potential as tools to investigate the role of the cholinergic system in AD.},
  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{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}
}