@phdthesis{Volpato2021,
  author    = {Volpato, Daniela},
  title     = {Bitopic Ligands and their molecular fragments for the study of the M1 Muscarinic Receptor},
  doi       = {10.25972/OPUS-24881},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-248815},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The past decades have witnessed the development of new pharmaceutical compounds that modulate receptor function by targeting allosteric sites. Allosteric sites are, by definition, domains topographically distinct from the orthosteric binding pocket where the natural ligand binds. Exploring the possibilities of linking orthosteric and allosteric pharmacophores in one compound to yield 'bitopic' compounds is a strategy derived from the "message-address" concept by Schwyzer , first applied to GPCRs by Portoghese et al. This concept explicitly underlines the orthosteric/allosteric combination, in opposite to the more general umbrella term bivalent. The broad possibilities of bitopic ligands in the pharmaceutical field are under continuous study. Bitopic compounds are promising pharmaceutical tools for taking advantage of the allosteric binding to achieve subtype selectivity while preserving high affinity at the receptor. The development of bitopic ligands, based on the idea of combining high affinity (via orthosteric sites) with high selectivity (via allosteric sites), have led to the development of highly selective bivalent ligands for GPCRs , such as for the opioid receptors , muscarinic acetylcholine receptors (mAChRs), serotonin receptors, cannabinoid receptors, and gonadotropin-releasing hormone receptors. This concept has even been extended to other receptors, for examples nicotinic receptors and other proteins, such as acetylcholinesterases and the tyrosine kinase receptors TrkA and TrkC. The reasons to pursue a bitopic ligand approach are various. An improved affinity for the target GPCR and/or an improved selectivity either at the level of receptor subtype, or at the level of signaling pathway. Another advantage of bitopic ligands over purely allosteric ligands is that the former rely on the appropriate presence of endogenous agonist tone to mediate their effects, whereas a bitopic ligand would engage the orthosteric site irrespective of the presence or absence of endogenous tone. By way of introduction to the hybrid approach, a review of the concept of hybrids compounds targeting the cholinergic system is presented in section A of this thesis. Recent updates in hybrid molecule design as a strategy for selectively addressing multiple target proteins involved in Alzheimer's disease (AD) is here reported . This represents the potential and the growing interest in hybrid compound as pharmacological tools to achieve receptor subtype selectivity and/or, to study the overall functional activity of the receptor. Until now, muscarinic acetylcholine receptors (mAChRs) have proved to be a particularly fruitful receptor model for the development and characterization of bitopic ligands. In this thesis, several examples of new muscarinic bitopic approach are reported in the results section. A study of bipharmacophoric ligands composed of the muscarinic positive allosteric modulators (BQCAderived compounds) linked with chain of various lengths to different orthosteric building blocks is reported in the result part 1. Synthesis and examination of the potential pharmacological characteristic of Oxotremorine-BQCAd compounds and Xanomeline-BQCAd hybrid derivatives are described in results parts 2 and 4, respectively. Moreover, the bitopic concept has even been extended to other proteins, such as acetylcholinesterase. In the result part 5 an overview of the new Tacrine-Xanomeline hybrids aiming to improve the inhibitory potency of the acetylcholinesterase and simultaneously to increase the cholinergic tone, via the xanomelinic portion acting on the M1 receptor is given. A new trivalent approach is presented for the first time to deepen the study of the M1 muscarinic receptor in the result part 6. Moreover, the synthesis of a new series of iperoxo-derived alkane, bis(ammonio)alkane-type and rigidified chain ligands is given in the result part 7 together with some prospects for further research.},
  subject      = {Ligand <Biochemie>},
  language  = {en}
}
@phdthesis{Unger2020,
  author    = {Unger, Nina},
  title     = {Stability of Tryptophan in Parenteral Amino Acid Solutions: Identification of Degradation Products and Development of HPLC Analysis Methods},
  doi       = {10.25972/OPUS-19982},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-199825},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {The stability of Trp in pure solutions and in parenteral AA formulations was evaluated with regard to typically used manufacturing processes, storage conditions and primary packaging. Therefore, thorough stability studies on Trp solutions were conducted beforehand. The applied stressing method, i.e. steam sterilization by autoclave, are chemically seen relatively mild but showed to be efficient to induce Trp degradation in the presence of oxygen. Subsequent identification, separation and characterization were challenging due to similar substance properties, numerous stereoisomers and pairs of diastereomers found amongst them. However, the identified o-aminoacetophenone compounds, Kyn and NFK, are associated with photo reactivity and have photo-oxidizing properties. Thus, best possible protection from UV-light, together with strict oxygen expulsion, are the most important criteria to impede Trp degradation after autoclaving. The identification of Trp degradation products was assisted by the compilation of a substance library, which included manifold reported and chemically plausible Trp degradation substances. The substances were classified for priority and their early or late-stage occurrence. The large number of possible substances and stereoisomers was narrowed down with the information retrieved from LC-UV/MS experiments. However, final identification was achieved by the synthesis of proposed substances as references. The following eight substances were characterized as Trp degradation substances: Kyn, NFK and three pairs of diastereomers R,R/R,S DiOia, R,R/R,S Oia and cis/trans PIC. Fig. 33 shows the proposed degradation pathway and demonstrates the close chemical relationship, which may be an explanation for the conversion of some substances into each other during the storage period. The proposed pathway brings together the results of different Trp stability and stressing studies, respectively [89, 94, 97, 98, 103, 133]. To our knowledge, the simultaneous formation of the identified degradation substances has not been reported before and especially not under the stressing conditions applied. The application of a traditional RP-HPLC method was compared to two developed IP-HPLC methods and a RP-HPLC methods using a modified perfluorinated column. Orthogonal analyses methods and especially the combination of UV and MS detection are necessary in order to indicate potentially undetected degradation substances. Main evaluation criteria were the separation performance, analyses time, reproducibility and feasibility. The best results upon assessment of all Trp degradation products, in both; pure Trp solutions and pharmaceutical formulations, were obtained by a traditional RP-HPLC. The optimized method was validated according to ICH guidelines Q2(R1) and meets the criteria of a stability-indicating HPLC-UV method. The validated method has a sufficient separation performance with an adequate selectivity indicating the Trp degradation substances next to each other and next to other AAs in finished pharmaceutical formulations. The detailed knowledge of Trp degradation and the method presented may be transferred practically to the pharmaceutical industry processing Trp-containing products. In general, the findings might contribute to the quality management of such pharmaceutical products during manufacturing and storage. Additionally, the study results provide basic information for the establishment of an impurity consideration following the ICH guidelines Q3B (R2) (impurities in new drug products) for products containing Trp. However, further development of the method applying more sophisticated detectors or more potent HPLC techniques like e.g. UHPLC and the implication of more sensitive (MS) detectors like ToF-MS would be advantageous with regard to economic and practical aspects.},
  subject      = {Stabilit{\"a}t},
  language  = {en}
}
@phdthesis{Geyer2023,
  author    = {Geyer, Florian},
  title     = {Targeting of M\(_2\) and M\(_4\) Muscarinic Receptor Subtypes with New Dualsteric Ligands},
  doi       = {10.25972/OPUS-27150},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-271506},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {As part of the parasympathetic nervous system, muscarinic receptors are involved in the regulation of numerous functions in the human body. However, targeting a specific subtype of muscarinic receptors is challenging due to the high degree of similarity within the binding site of the endogenous neurotransmitter acetylcholine. Therefore, this study focused on the investigation of dualsteric ligands. Such hybrid ligands target the orthosteric acetylcholine binding site and, simultaneously, a distinct allosteric binding site. Since allosteric binding regions show significant structural differences throughout muscarinic receptor subtypes, it was aimed to produce selective ligands by means of combination of two pharmacophores in one molecule. Herein, the thienopyridine derivatives LY2033298 and LY2119620 were chosen as allosteric moieties. Based on literature studies, the investigated allosteric modulators were analyzed in terms of adequate attachment points for the combination with an orthosteric agonist. As orthosteric units, muscarinic superagonist iperoxo, xanomeline, and TMA were applied in this work. Since the distance between orthosteric and allosteric moieties plays a crucial role for dualsteric ligand binding, the linker chain length was also varied. Pharmacological investigations of the synthesized hybrid ligands were perfomed via FRET- and BRET-assay measurements.},
  subject      = {GTP-bindende Proteine},
  language  = {en}
}
@phdthesis{Volpp2021,
  author    = {Volpp, Miriam},
  title     = {Bestimmung der Plasmaproteinbindung von niedrig affinen Liganden am Beispiel der Ephedra-Alkaloide},
  doi       = {10.25972/OPUS-21961},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-219619},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Zur Bestimmung der Bindungsaffinit{\"a}t von Liganden zu den Plasmaproteinen, insbesondere Albumin, wurden {\"u}ber die Jahre zahlreiche Methoden entwickelt. Die Grundlage dieser Arbeit war die Bestimmung der Plasmaproteinbindung der Ephedra-Alkaloide unter Verwendung einzelner dieser etablierten Methoden. Aufgrund ihres Anwendungsgebiets als Notfallmedikation bei An{\"a}sthesie-bedingter Hypotonie und den damit verbundenen Anforderungen an die Pharmakokinetik, sollten die Ephedra-Alkaloide niedrig-affine Liganden der Plasmaproteine darstellen. In der Literatur und in vorhergehenden Arbeiten wurden f{\"u}r die Ephedra-Alkaloide jedoch sehr unterschiedliche, teilweise der Indikation widersprechende Affinit{\"a}ten bestimmt. Daher sollte im Rahmen dieser Arbeit das Ausmaß der Plasmaproteinbindung der Ephedra-Alkaloide weiter untersucht und die Affinit{\"a}t zu Albumin bzw. anderen Plasmaproteinen im humanen Serum bestimmt werden. Neben der Affinit{\"a}t sollte auch die Stereoselektivit{\"a}t der Bindung genauer betrachtet werden, die bei der Bindung vieler Wirkstoffe eine Rolle spielt. Als Referenzmethode diente die kontinuierliche Ultrafiltration, die auch schon bei H{\"o}rst verwendet wurde. Folgende Schlussfolgerungen konnten aus den Ergebnissen dieser Arbeit gezogen werden: 1) Die Ergebnisse der kontinuierlichen Ultrafiltration zeigten, dass die Ephedra-Alkaloide, Ephedrin und Pseudoephedrin, ein nur geringes Ausmaß an Plasmaprotein-bindung von 4 - 9 \% gegen{\"u}ber bovinem und humanem Serumalbumin zeigen. Eine deutlich h{\"o}here Plasmaproteinbindung von 19 - 37 \% konnte hingegen bei der Verwendung von humanem Serum bestimmt werden. Die Affinit{\"a}t von Pseudoephedrin war dabei jeweils geringer als die von Ephedrin. 2) Diese Ergebnisse mit humanem Serum und die Tatsache, dass Albumin vorwiegend saure Stoffe bindet, legen nahe, dass die Ephedra-Alkaloide vermehrt an andere Plasmaproteine in Serum binden. Erste Messergebnisse mit saurem α1 Glykoprotein best{\"a}tigen diese Vermutung. 3) Eine Stereoselektivit{\"a}t konnte nur in geringem Maß bei (+) Ephedrin beobachtet werden, wobei der Unterschied nur im Serum signifikant ist. Pseudoephedrin dagegen zeigte keinerlei Stereoselektivit{\"a}t. Diese Beobachtung passt zu den Schlussfolgerungen der Pfeiffer'schen Regel zur Stereoselektivit{\"a}t einer Bindung. 4) Andere Sympathomimetika mit einer zus{\"a}tzlichen Phenolgruppe im Molek{\"u}l zeigen eine {\"a}hnlich niedrige Affinit{\"a}t zu Albumin von ca. 10 \%. Eine zus{\"a}tzliche Phenolgruppe scheint die sauren Eigenschaften des Liganden nicht ausreichend zu erh{\"o}hen, um die Affinit{\"a}t zu Albumin signifikant zu steigern. 5) Das terti{\"a}re Kohlenstoffatom am Stickstoff des Ephedrins scheint in gewisser Weise an der Bindung zu Albumin beteiligt zu sein. Sympathomimetika mit einer zus{\"a}tzlichen Methylgruppe an diesem Kohlenstoffatom, wie Ephedrin, Pseudoephedrin und Oxilofrin, zeigen eine gr{\"o}ßere Streuung der Messergebnisse. Eine zus{\"a}tzliche Methylgruppe in dieser Position scheint die Bindung daher sterisch zu hindern. 6) Die Ergebnisse der diskontinuierlichen Ultrafiltration best{\"a}tigen weitestgehend die Ergebnisse der kontinuierlichen Ultrafiltration 7) Eine Bestimmung des Ausmaßes der Plasmaproteinbindung von niedrig-affinen Stoffen ist mit den anderen orthogonalen Methoden ACE, NMR und iTC nicht m{\"o}glich. Diese drei verwendeten Methoden trennen nicht wie die klassischen Methoden den gebundenen vom ungebundenen Wirkstoff, sondern beruhen auf einer Ver{\"a}nderung bestimmter Messparameter: bei der ACE die Migrationszeit, bei der NMR-Spektroskopie die chemische Verschiebung der Signale bzw. der Diffusionskoeffizient und bei der iTC die frei werdende Bindungsw{\"a}rme. Bei allen drei Methoden war die {\"A}nderung der Messgr{\"o}ße aufgrund der niedrigen Plasmaproteinbindung zu gering, um auswertbar zu sein. 8) Eine St{\"o}rgr{\"o}ße bei die orthogonalen Methoden war vielfach auch das Albumin selbst bzw. dessen Eigenschaften. Bei der Affinit{\"a}ts-Kapillarelektrophorese sind physiologische HSA-Konzentrationen wegen des starken Basislinienrauschens nicht messbar. Zudem bewirkt der Albuminzusatz im Trennpuffer eine Viskosit{\"a}ts{\"a}nderung, die den EOF verlangsamt und so die Messung st{\"o}rt. Bei der NMR-Spektroskopie k{\"o}nnen wegen der {\"U}berlagerung der Signale durch die breiten Albuminbanden weder Ver{\"a}nderungen in der chemischen Verschiebung noch des Diffusionskoeffizienten zuverl{\"a}ssig bestimmt werden. In der iTC erschwerte die Schaumbildung der L{\"o}sung, die durch die Oberfl{\"a}chenaktivit{\"a}t des Albumins verursacht wird, die Messung. In dieser Arbeit konnte somit das Ausmaß der Plasmaproteinbindung der Ephedra-Alkaloide mit verschiedenen Methoden erfolgreich bestimmt werden. Damit best{\"a}tigte diese Arbeit, dass die Ephedra-Alkaloide, wie deren Indikation vermuten l{\"a}sst, zu den niedrig affinen Liganden des Albumins z{\"a}hlen. Um genauer eingrenzen zu k{\"o}nnen durch welche Plasmaproteine im Blutserum die Ephedra-Alkaloide transportiert werden, sollten die Untersuchungen zum sauren α1-Glykoprotein fortgesetzt und gegebenenfalls durch weitere Bestimmungen mit anderen Plasmaproteinen erg{\"a}nzt werden. Die Ergebnisse dieser Arbeit haben auch gezeigt, dass viele der unz{\"a}hligen Methoden zur Untersuchung der Plasmaproteinbindung bei der Bestimmung von niedrig affinen Liganden ihre Grenzen haben. Nach wie vor sind zur Bestimmung einer niedrigen Bindungsaffinit{\"a}t weiterhin die klassischen Methoden, wie die kontinuierliche Ultrafiltration, Mittel der Wahl. Nicht zuletzt deshalb erfreuen sich diese Methoden auch heute noch großer Beliebtheit.},
  subject      = {Proteinbindung},
  language  = {de}
}
@phdthesis{Walther2023,
  author    = {Walther, Rasmus},
  title     = {Analysis of weakly chromophore impurities by means of liquid chromatography coupled with charged aerosol detection and mass spectrometry},
  doi       = {10.25972/OPUS-32186},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-321862},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {In all the projects presented, it is evident that the selection of suitable separation conditions is only one side of the coin. Equally crucial in the development of methods for the quality assessment of APIs/drugs is the right detection system. The application of CAD as an alternative to UV detection at low wavelength of the two weak chromophore main degradation products of the very polar, zwitterionic API carbocisteine requires the volatility of the mobile phase. Therefore, as a substitute for the non-volatile ion pairing reagent tetrabutylammonium hydroxide (TBAOH), six different volatile alkylamines as well as a RP/SAX mixed-mode column were evaluated. The best selectivity and separation performance comparable to TBAOH was achieved with the RP/SAX column and a mixture of formic acid and trifluoroacetic acid. For the simultaneous optimisation of the evaporation temperature of the CAD as a function of two chromatographic parameters, a central composite design was chosen and the "desirability function" was subsequently applied for modelling. In addition, column bleeding was investigated with a second RP/SAX column (different batch) with the result that the acetonitrile percentage had to be adjusted and preconditioning by injection of concentrated samples is essential. The final mixed-mode method was finally validated with both columns according to the ICH Q2 (R1) guideline. Based on this, an MS-compatible method was developed with little effort using an identical RP/SAX column in UPLC dimension for the untargeted analysis by HRMS of two carbocisteine-containing prototype syrup formulations. For a comprehensive characterisation, HRMS and MS/HRMS data were recorded simultaneously by information dependent acquisition mode. Based on the exact masses, isotope patterns and an in silico plausibility check of the fragment spectra, the prediction of the structures of the unknown impurities was possible. In both syrup samples, which had been stored for nine months at 40 °C and 75 \% r.h., two additional impurities of carbocisteine (i.e. lactam of the sulfoxides and disulphide between cysteine and thioglycolic acid) were identified by comparison with the corresponding prototype placebo samples using general unknown comparative screening. In addition, the formation of Maillard products by binary mixtures with 13C-labelled sugars was revealed in the sucrose-containing formulation. For the promising hyphenation of the UV detector with the CAD for the simultaneous detection of all UV-active impurities of the cholesterol-lowering drug simvastatin and the only weak chromophore dihydrosimvastatin, the Ph. Eur. method had to be adapted. Besides replacing phosphoric acid with trifluoroacetic acid, the gradient also had to be adjusted and a third critical peak pair was observed. Based on validation experiments (according to the ICH Q2 (R1) guideline), the suitability of the CAD for sensitive detection (LOQ = 0.0175 \% m/m) was proven.  To further investigate the robustness of the adapted method and CAD, a Plackett-Burman design was chosen. None of the factors had a statistically significant effect on the S/N of the CAD in the ranges tested. Regarding the three critical peak pairs, on the other hand, the factors to be controlled were statistically established, so that a targeted correction is possible if the system suitability test is not passed. The idea of employing a hyphenated UV-CAD system was finally applied to the structurally closely related lovastatin and its specified impurity dihydrolovastatin. Here, the CAD showed a significantly better S/N compared to the compendial UV detection at 200 nm. The suitability of CAD for the analysis of non-volatile fatty acids in polysorbate 80 (PS80) as favourable alternative to the Ph. Eur. GC method (no time-consuming, error-prone and toxic derivatisation) has already been demonstrated. The aim of this project was therefore to develop a robust method with a focus on the AQbD principles, which can be used for the analysis of other excipients with similar fatty acid composition. After the definition of the analytical target profile and a risk assessment by means of an Ishikawa diagram, a suitable C18 column and the chromatographic framework conditions (formic acid concentration and initial/final gradient conditions) were selected after only few preliminary runs. The remaining critical method parameters were then investigated with the help of DoE and RSM. Using the obtained model equations, Monte Carlo simulations were performed to create the method operable design region as a region of theoretical robustness. After validation according to ICH Q2 (R1), the fatty acid composition of a magnesium stearate batch was successfully analysed as a further application example in addition to PS80. The CAD was able to prove its potential in all the issues investigated in the context of this doctoral thesis. As a cost-effective alternative compared to MS instruments, it thus closes a gap in the quality assessment of APIs or excipients without a suitable chromophore. The easy method transfer to (HR)MS instruments also allows for a unique degree of sample characterisation through untargeted approaches in case of new impurities. For resource- and time-efficient work, the possibilities and limitations of software tools for method development and data evaluation as well as the application of risk-based approaches such as AQbD should also be considered.},
  subject      = {Carbocistein},
  language  = {en}
}
@phdthesis{Heinz2023,
  author    = {Heinz, Christine Silvia},
  title     = {Synthesis of Analogues and Hybrid Ligands of Pilocarpine for the Study of Muscarinic Receptor Dynamics},
  doi       = {10.25972/OPUS-28148},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-281486},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Muscarinic acetylcholine receptors (mAChRs) are involved in signal transmission at the synapses of the parasympathetic nervous system. The five subtypes of mAChRs regulate various body functions such as heart function, gland secretion, memory, and learning. For the development of drugs with the least side-effects possible, the molecular causes of subtype selectivity and signalling bias are under investigation. In this context, the study of dualsteric ligands binding simultaneously to the orthosteric and the allosteric binding sites of the receptor is of high interest. To date, dualsteric ligands were synthesised as hybrids of full agonists or superagonists being the orthosteric element, linked to known subtype selective allosteric fragments. In this work, the existing library was expanded to hybrid ligands based on the partial agonist pilocarpine. A suitable linker attachment point to pilocarpine was investigated. For this aim, pilocarpine (2), isopilocarpine (15), pilosinine (16) and desmethyl pilosinine (35) were synthesised as orthosteric ligands and orthosteric fragments for the construction of the hybrid molecules (Figure 42). Pilocarpine was liberated from the commercial hydrochloride or nitrate salt and isopilocarpine was generated by epimerisation of pilocarpine. Pilosinine was synthesised in a Michael addition reaction of a dithiane carrying the imidazole moiety 82 onto the lactone precursor furan-2(5H)-one (83) followed by complete deprotection (Figure 43a).[133] The desmethyl pilosinine (35) was obtained in a newly developed synthetic route based on a Horner-Wadsworth-Emmons (HWE) reaction to build the methylene bridge between the imidazole aldehyde and the precursor of the lactone moiety 57 (Figure 43b). All four orthosters were converted to the respective dualsteric compounds with a naphmethonium fragment as allosteric moiety. The four orthosteric fragments and the four hybrid molecules with a linker length of six methylene units were tested for their dose dependent G protein recruitment at the receptor subtypes M1-5 using a mini-G nanoBRET assay. The study of the orthosteric ligands revealed that pilocarpine has the highest ability of all four orthosters to induce activity at all receptor subtypes. A change of the cis- to a trans-configuration of the lactone substituents or a complete removal of the ethyl substituent provoked a significant reduction of activity. Removal of the methyl substituent of the imidazole moiety led to improved receptor activation. The efficacies of the hybrid ligands show that the linker attachment at the imidazole moiety of pilocarpine and its analogues does not abolish activity and hybrid formation of isopilocarpine even improved receptor activation. Thus, the linker attachment point seems a valid choice, but linker length might not be optimum. In contrast to the orthosters, the trans-substitution of the lactone was advantageous for receptor activation of the hybrid ligands. The hybrid without a methyl substituent at the imidazole (69) had an increased efficacy. Additionally, the naphmethonium fragment lowered the maximum effect of pilocarpine, whereas the activity of isopilocarpine was increased. The intensity of both effects was influenced by the subtype selectivity produced by naphmethonium leading, in the case of the pilocarpine hybrid, to less decreased responses or, in the case of the isopilocarpine hybrid, to more increased responses at the M2 and M4 receptors. The results generally lead to the assumption that the allosteric moiety strongly influences the binding poses of the hybrid ligands so that the orthosteric fragments do not interact with the binding site in the same way as the orthosters alone. A second project was based on molecular dynamics simulations of the binding pose of pilocarpine,[73] leading to the hypothesis that the partial agonism of pilocarpine results from an equilibrium between an agonistic and an antagonistic binding pose at the orthosteric binding site of the receptor. The ratio of occupancy of both binding poses determines the observed efficacy of pilocarpine. The orthosteric binding site provides more space for the ethyl substituent in the supposed antagonistic pose than in the agonistic binding pose. This hypothesis was tested by the synthesis and pharmacological evaluation of pilocarpine analogues with alkyl substituents of different sizes at the lactone (16, 31a, c, d) (Figure 44). The analogues with larger alkyl residues are expected to shift the equilibrium towards the antagonistic binding pose, the analogues with smaller residues should have the inverse effect. The synthesis of the pilocarpine analogues was first attempted as a mixture of stereoisomers which were supposed to be separated at the end of the synthetic route. The racemic mixture of the thermodynamically more stable trans-isomers of the target compounds was prepared in a one-pot Michael-addition-alkylation reaction of a dithiane imidazole onto furan-2(5H)-one similarly to the synthesis of pilosinine (Figure 45). The resulting enolate was quenched by an iodoalkane to achieve alkylation of the lactone and subsequent complete deprotection yielded the racemic trans-analogues of pilocarpine.[133] After unsuccessful attempts of chiral resolution, the mixture of trans-isomers was converted to a mixture of all four possible diastereomers in a kinetic epimerisation reaction.[95] A separation of the stereoisomers was not possible in this project so only the racemic molecule 16 (pilosinine, R = H) was obtained from this synthetic route. For the selective synthesis of the cis-isomers following a patent from Reimann,[146] both stereocenters of the target molecules were produced in the last synthetic step by a syn-hydrogenation of the α,β-unsaturated precursor (Figure 46). The racemic pilocarpine analogues, except the butyl derivative (31d), were purified by crystallisation as their nitrate salts. This provided the racemic mixtures with less than 8\% of the trans-isomers as impurity. The racemic pilocarpine (2), itself, was obtained with 15\% trans-impurity and was used as reference compound. Additionally, the possibility of chiral resolution by chromatographic methods was demonstrated in the case of the methyl derivative (31a). The pharmacological testing of the desired enantiomer of 31a is in progress.},
  subject      = {Muskarinrezeptor},
  language  = {en}
}
@phdthesis{Scheuplein2023,
  author    = {Scheuplein, Nicolas Julian},
  title     = {Synthesis and Characterization of Antimicrobial Inhibitors of the "Macrophage Infectivity Potentiator" Protein and Fluorescent Probes},
  doi       = {10.25972/OPUS-32189},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-321892},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {This dissertation focuses on Mip (macrophage infectivity potentiator protein) inhibitors in response to increasing antibiotic resistance. The study follows an antivirulence approach, which aims to inhibit the non-essential Mip protein without exerting too much selective pressure. Three focus areas were (1) development and synthesis of a fluorescent probe for screening Mip inhibitors via fluorescence polarization; (2) design and synthesis of broad spectrum Mip inhibitors bearing a side chain; and (3) understanding the metabolism of Mip inhibitors and identification of active metabolites. A sub-study addressed the biotinylation of anti-leishmanial compounds from Valeriana wallichii rhizomes, with three tracer molecules synthesized for future pull-down experiments.},
  subject      = {Antibiotikum},
  language  = {en}
}
@phdthesis{Schmidt2023,
  author    = {Schmidt, Sebastian},
  title     = {A closer look at long-established drugs: enantioselective protein binding and stability studies},
  doi       = {10.25972/OPUS-34594},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-345945},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {The aim of this work was to investigate older, established drugs. The extent of the protein binding of chiral ephedra alkaloids to AGP and of ketamine to albumin was determined. Since enantiomers of these drugs are individual available, the focus was on possible enantioselective binding and structural moieties involved in the binding. Previously published work suggested that ephedrine and pseudoephedrine can bind stereoselectively to proteins other than albumin in serum. For the determination of the extent of protein binding, the established ultrafiltration with subsequent chiral CE analysis was used. To determine the influence of basicity on binding, the drugs methylephedrine and norephedrine were also analyzed. Drug binding to AGP increased with increasing basicity as follows: norephedrine < methylephedrine < ephedrine < pseudoephedrine. pKaff was determined both graphically using the Klotz plot and mathematical indicating a low affinity of the ephedra alkaloids to AGP. Using STD-NMR spectroscopy experiments the aromatic protons and the C-CH3 side chain were shown to be most strongly involved in binding, which could be confirmed by molecular docking experiments in more detail. For all drugs, van der Waals-, π π , cationic interactions, hydrogen bonds, and a formation of a salt bridge were observed. The individual enantiomers showed no significant differences and thus the binding of ephedra alkaloids to AGP is not significant. In contrast to the ephedra alkaloids, the possible enantioselective binding to albumin was investigated for R and S ketamine. Again, ultrafiltration followed by CE analysis was performed. The binding of ketamine to one main binding site could be identified. A non-linear fit was used for the determination of pKaff. Using the NMR methods STD-NMR, waterLOGSY-NMR, and CPMG-NMRspectroscopy: the aromatic protons as well as the protons of the NCH3 methyl group showed the largest signal intensity changes, while the cyclohexanone protons showed the smallest changes. pKaff was also determined by the change in the chemical shift at different drug-protein ratios. These obtained values confirm the values obtained from ultrafiltration. Based on this, ketamine is classified as a low-affinity ligand to albumin. There were no significant differences between the individual enantiomers and thus the binding of ketamine to albumin is not a stereoselective process. Using statistical design of experiments an efficient chiral CE method for determining the extent of protein binding of R and S ketamine to albumin was developed and validated according to ICH Q2 (R1) guideline. The stability of ketamine was also investigated because a yellowish discoloration of an aqueous solution of ketamine developed under heat. XRPD investigations showed the same crystal structure for all batches examined. An untargeted screening using LC HRMS as well as LC UV measurements showed no degradation of ketamine or the presence of impurities in stress and non-stressed ketamine solutions, confirming the stability of ketamine under the stress conditions investigated. The lower the quality of the water used in the stress tests, the more intense the yellow discoloration occurred. The impurity or the mechanism that causes the yellow discoloration could not be identified.},
  subject      = {Proteinbindung},
  language  = {en}
}
@phdthesis{Weinmann2023,
  author    = {Weinmann, Joshua},
  title     = {Chemical Modifications of Quinolone Amides Against African Trypanosomiasis: Balancing Solubility, Bioactivity, and Cytotoxicity},
  doi       = {10.25972/OPUS-29659},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-296599},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {The human African trypanosomiasis is a neglected tropical disease, which is caused by the protozoan Trypanosoma brucei and transmitted by the bite of the tsetse fly. An untreated infection leads to death. However, only a few drugs with significant drawbacks are currently available for treatment. In this thesis, quinolone amides with an antitrypanosomal activity were synthesized and their biological and physicochemical properties were measured. New structure-activity relationships and a promising lead structure were discovered.},
  subject      = {Trypanosomiase},
  language  = {en}
}
@phdthesis{Schlauersbach2023,
  author    = {Schlauersbach, Jonas},
  title     = {The bile-drug-excipient interplay},
  doi       = {10.25972/OPUS-29653},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-296537},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {The bile system in vertebrates is an evolutionary conserved endogenous solubilization system for hydrophobic fats and poorly water-soluble vitamins. Bile pours out from the gallbladder through the common bile duct into the duodenum triggered by cholecystokinin. Cholecystokinin is released from enteroendocrine cells after food intake. The small intestine is also the absorption site of many orally administered drugs. Most emerging drug candidates belong to the class of poorly water-soluble drugs (PWSDs). Like hydrophobic vitamins, these PWSDs might as well be solubilized by bile. Therefore, this natural system is of high interest for drug formulation strategies. Simulated intestinal fluids containing bile salts (e.g., taurocholate TC) and phospholipids (e.g., lecithin L) have been widely applied over the last decade to approximate the behavior of PWSDs in the intestine. Solubilization by bile can enhance the oral absorption of PWSDs being at least in part responsible for the positive "food effect". The dissolution rate of PWSDs can be also enhanced by the presence of bile. Furthermore, some PWSDs profit from supersaturation stabilization by bile salts. Some excipients solubilizing PWSDs seemed to be promising candidates for drug formulation when investigated in vitro without bile. When tested in vivo, these excipients reduced the bioavailability of drugs. However, these observations have been hardly examined on a molecular level and general links between bile interaction in vitro and bioavailability are still missing. This thesis investigated the interplay of bile, PWSDs, and excipients on a molecular level, providing formulation scientists a blueprint for rational formulation design taking bile/PWSD/excipient/ interaction into account. The first chapter focus on an in silico 1H nuclear magnetic resonance (NMR) spectroscopy-based algorithm for bile/drug interaction prediction. Chapter II to IV report the impact of excipients on bioavailability of PWSDs interacting with bile. At last, we summarized helpful in vitro methods for drug formulation excipient choice harnessing biopharmaceutic solubilization in chapter V. Chapter I applies 1H NMR studies with bile and drugs on a large scale for quantitative structure-property relationship analysis. 141 drugs were tested in simulated intestinal media by 1H NMR. Drug aryl-proton signal shifts were correlated to in silico calculated molecular 2D descriptors. The probability of a drug interacting with bile was dependent on its polarizability and lipophilicity, whereas interaction with lipids in simulated intestinal media components was dependent on molecular symmetry, lipophilicity, hydrogen bond acceptor capability, and aromaticity. The probability of a drug to interact with bile was predictive for a positive food effect. This algorithm might help in the future to identify a bile and lipid interacting drug a priori. Chapter II investigates the impact of excipients on bile and free drug fraction. Three different interaction patterns for excipients were observed. The first pattern defined excipients that interacted with bile and irreversibly bound bile. Therefore, the free drug fraction of bile interacting drugs increased. The second pattern categorized excipients that formed new colloidal entities with bile which had a high affinity to bile interacting drugs. These colloids trapped the drug and decreased the free drug fraction. The last excipient pattern described excipients that formed supramolecular structures in coexistence with bile and had no impact on the free drug fraction. These effects were only observed for drugs interacting with bile (Perphenazine and Imatinib). Metoprolol's free drug fraction, a compound not interacting with bile, was unaffected by bile or bile/excipient interaction. We hypothesized that bile/excipient interactions may reduce the bioavailability of bile interacting drugs. Chapter III addresses the hypothesis from chapter II. A pharmacokinetic study in rats revealed that the absorption of Perphenazine was reduced by bile interacting excipients due to bile/excipient interaction. The simultaneous administration of excipient patterns I and II did not further reduce or enhance Perphenazine absorption. Conversely, the absorption of Metoprolol was not impacted by excipients. This reinforced the hypothesis, that drugs interacting with bile should not be formulated with excipients also interacting with bile. Chapter IV further elaborates which in vitro methods using simulated intestinal fluids are predictive for a drug's pharmacokinetic profile. The PWSD Naporafenib was analyzed in vitro with simulated intestinal fluids and in presence of excipients regarding solubility, supersaturation, and free drug fraction. Naporafenib showed a strong interaction with TC/L from simulated bile. Assays with TC/L, but not without identified one excipient as possibly bioavailability reducing, one as supersaturation destabilizing, and the last as bile not interacting and supersaturation stabilizing excipient. A pharmacokinetic study in beagle dogs outlined and confirmed the in vitro predictions. The Appendix summarizes in vivo predictive methods as presented in chapter I to IV and rationalizes experimental design paving the way towards a biopharmaceutic excipient screening. The first presented preliminary decision tree is transformed into a step-by-step instruction. The presented decision matrix might serve as a blueprint for processes in early phase drug formulation development. In summary, this thesis describes how a drug can be defined as bile interacting or non-interacting and gives a guide as well how to rate the impact of excipients on bile. We showed in two in vivo studies that bile/excipient interaction reduced the bioavailability of bile interacting drugs, while bile non-interacting drugs were not affected. We pointed out that the bile solubilization system must be incorporated during drug formulation design. Simulated gastrointestinal fluids offer a well-established platform studying the fate of drugs and excipients in vivo. Therefore, rational implementation of biopharmaceutic drug and excipient screening steers towards efficacy of oral PWSD formulation design.},
  subject      = {Solubilisation},
  language  = {en}
}