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Chronic inflammatory diseases such as rheumatoid arthritis, type 2 diabetes and cardiovascular diseases, are associated with the homeostatic imbalance of one of several physiological systems combined with the lack of spontaneous remission, which causes the disease to persevere throughout patients’ lives. The inflammatory response relies mainly on tissue-resident, pro-inflammatory M1 type macrophages and, consequently, a chance for therapeutic intervention lies in driving macrophage polarization towards the anti-inflammatory M2 phenotype. Therefore, anti-inflammatory cytokines that promote M2 polarization, including interleukin-4 (IL4), have promising therapeutic potential. Unfortunately, their systemic use is hampered by a short serum half-life and dose-limiting toxicity. On the way towards cytokine therapies with superior safety and efficacy, this thesis is focused on designing bioresponsive delivery systems for the anti-inflammatory cytokine IL4.
Chapter 1 describes how anti-inflammatory cytokines are tightly regulated in chronic, systemic inflammation as in rheumatoid arthritis but also in acute, local inflammation as in myocardial infarction. Both diseases show a characteristic progression during which anti-inflammatory cytokine delivery is of variable benefit. A conventional, passive drug delivery system is unlikely to release the cytokines such that the delivery matches the dynamic course of the (patho-)physiological progress. This chapter presents a blueprint for active drug delivery systems equipped with a 24/7 inflammation detector that continuously senses for matrix metalloproteinases (MMP) as surrogate markers of the disease progress and responds by releasing cytokines into the affected tissues at the right time and place. Because they are silent during phases of low disease activity, bioresponsive depots could be used to treat patients in asymptomatic states, as a preventive measure. The drug delivery system only gets activated during flares of inflammation, which are then immediately suppressed by the released cytokine drug and could prevent the steady damage of subclinical chronic inflammation, and therefore reduce hospitalization rates.
In a first proof of concept study on controlled cytokine delivery (chapter 2), we developed IL4-decorated particles aiming at sustained and localized cytokine activity. Genetic code expansion was deployed to generate muteins with the IL4’s lysine 42 replaced by two different unnatural amino acids bearing a side chain suitable for click chemistry modification. The new IL4 muteins were thoroughly characterized to ensure proper folding and full bioactivity. Both muteins showed cell-stimulating ability and binding affinity to IL4 receptor alpha similar to those of wild type IL4. Copper-catalyzed (CuAAC) and strain-promoted (SPAAC) azide–alkyne cycloadditions were used to site-selectively anchor IL4 to agarose particles. These particles had sustained IL4 activity, as demonstrated by the induction of TF-1 cell proliferation and anti-inflammatory M2 polarization of M-CSF-generated human macrophages. This approach of site-directed IL4 anchoring on particles demonstrates that cytokine-functionalized particles can provide sustained and spatially controlled immune-modulating stimuli.
The idea of a 24/7 sensing, MMP driven cytokine delivery system, as described in the introductory chapter, was applied in chapter 3. There, we simulated the natural process of cytokine storage in the extracellular matrix (ECM) by using an injectable solution of IL4 for depot formation by enzyme-catalyzed covalent attachment to ECM components such as fibronectin. The immobilized construct is meant to be cleaved from the ECM by matrix-metalloproteinases (MMPs) which are upregulated during flares of inflammation. These two functionalities are facilitated by a peptide containing two sequences: a protease-sensitive peptide linker (PSL) for MMP cleavage and a sequence for covalent attachment by activated human transglutaminase FXIIIa (TGase) included in the injection mix for co-administration. This peptide was site-selectively conjugated to the unnatural amino acid at IL4 position 42 allowing to preserve wild type bioactivity of IL4. In vitro experiments confirmed the anticipated MMP response towards the PSL and TGase-mediated construct attachment to fibronectin of the ECM. Furthermore, the IL4-peptide conjugates were able to reduce inflammation and protect non-load bearing cartilage along with the anterior cruciate ligament from degradation in an osteoarthritis model in rabbits. This represents the first step towards a minimally invasive treatment option using bioresponsive cytokine depots with potential clinical value for inflammatory conditions.
One of the challenges with this approach was the production of the cytokine conjugate, with incorporation of the unnatural amino acid into IL4 being the main bottleneck. Therefore, in chapter 4, we designed a simplified version of this depot system by genetically fusing the bifunctional peptide via a flexible peptide spacer to murine IL4. While human IL4 loses its activity upon C-terminal elongation, murine IL4 is not affected by this modification. The produced murine IL4 fusion protein could be effectively bound to in vitro grown extracellular matrix in presence of TGase. Moreover, the protease-sensitive linker was selectively recognized and cleaved by MMPs, liberating intact and active IL4, although at a slower rate than expected. Murine IL4 offers the advantage to evaluate the bioresponsive cytokine depot in many available mouse models, which was so far not possible with human IL4 due to species selectivity.
For murine IL4, the approach was further extended to systemic delivery in chapter 5. To increase the half-life and specifically target disease sites, we engineered a murine IL4 variant conjugated with a folate-bearing PEG chain for targeting of activated macrophages. The bioactive IL4 conjugate had a high serum stability and the PEGylation increased the half-life to 4 h in vivo. Surprisingly, the folate moiety did not improve targeting in an antigen-induced arthritis (AIA) mouse model. IL4-PEG performed better in targeting the inflamed joint, while IL4-PEG-folate showed stronger accumulation in the liver. Fortunately, the modular nature of the IL4 conjugate facilitates convenient adaption of PEG chain length and the targeting moiety to further improve the half-life and localization of the cytokine.
In summary, this thesis describes a platform technology for the controlled release of cytokines in response to inflammation. By restricting the release of the therapeutic to the site of inflammation, the benefit-risk ratio of this potent class of biologics can be positively influenced. Future research will help to deepen our understanding of how to perfectly combine cytokine, protease-sensitive linker and immobilization tag or targeting moiety to tackle different diseases.
To diagnose diseases correctly requires not only trained and skilled personnel, but also cost-intensive and complex equipment. Rapid tests can help with the initial evaluation, but result generation can also take up to several hours, depending on the test system. At this point, novel bioresponsive diagnostic systems are used, responding to the disease related shift of biological processes. They monitor changes in the biological environment and can react to them e.g. with the release of substances. This can be used in drug delivery formulations but can also help to diagnose diseases occurring in the oral cavity and inform patients of their state of health. The tongue is herein used as a 24/7 available detector.
In section I of this work, the foundation for the development of these diagnostic systems was laid. A suitable flavoring agent was found, which is stable, can be coupled to the N-terminus of peptides and has a strongly conceivable taste. For the optimization of the protease-sensitive linker (PSL), an analytical system was established (PICS assay), which determines protease-specific cleavable amino acid sequences. In order to replace the PMMA particles previously required, an acetyl protecting group was introduced N-terminally as it protects peptides and proteins in the human body from degradation by human aminopeptidase. The new synthesized flavor was examined with a NIH cell line for cytotoxicity and with an electronic tongue setup for its bitterness.
Section II deals with the structure of a system which detects severe inflammations in the oral cavity, e.g. PA. The established PICS assay was used to confirm the previously used PSL sequence in its application. Using solid phase peptide synthesis, 3 linkers were synthesized which respond to the elevated MMP concentrations present in inflammation. The resulting peptides were acetylated and coupled with HATU/DIPEA to the modified denatonium. Cutting experiments with MMPs over different concentration and time ranges confirmed the response of the diagnostic sensor to these enzymes. The obtained construct was examined for cell toxicity by WST assay. The masked bitterness of the sensors was confirmed by an electronic tongue setup.
To address non-human proteases (and thereby infections), section III focuses on the establishment of detection system of a cysteine protease SpeB expressed by Streptococcus pyogenes. The in-house expression of SpeB using E. coli cells was established for this purpose. An analysis of the SpeB cleavage sites was performed using a PICS assay setup. Four constructs with different PSL were synthesized analogous to section II. Cleavage experiments with the expressed and purified SpeB showed a response of two constructs to the protease. In addition, a system was established to quantify the concentration of SpeB in human saliva using western blot technique with subsequent quantification.
In section IV a compound was synthesized which can now be coupled to a flavor. The final coupled construct is able to detect present NA activity specifically from influenza A and B. The market for existing influenza diagnostics was explored to determine the need for such a system. A neuraminic acid was modified in positions 4 and 7 and protected in such a way that subsequent coupling via the hydroxy-group in position 2 was selectively possible.
In summary, this results in a diagnostic platform that can be used anywhere, by anyone and at any time. This represents a new dimension in the rapid diagnosis of inflammations and bacterial or viral infections.
High-resolution nuclear magnetic resonance (NMR) spectroscopy is used in structure elucidation and qualitative as well as quantitative examination of product components. Despite the worldwide development of numerous innovative NMR spectroscopic methods, several official methods that analyze specific substances and do not represent a holistic analysis, are still in use for the quality control of drugs, food and chemicals. Thus, counterfeit or contaminated products of inferior quality can be brought onto the market and distributed despite previous quality controls. To prevent this, three NMR spectroscopic methods have been developed within the scope of this work (1) to study the peroxide value in vegetable and animal oils, (2) for the qualitative and quantitative analysis of metal cations and (3) to determine the enantiomeric excess in chiral alcohols. In oil analysis, titration methods are used to determine the bulk quality parameters such as peroxide value, which represents the concentration of peroxides. Titrations show several drawbacks, such as the need of a large amount of sample and solvents, cross reactions and the low robustness. Thus, an alternative NMR spectroscopic method was developed to improve the peroxide analysis by using triphenylphosphine as a derivatization reagent, which reacts with peroxides in a stoichiometric ratio of 1:1 forming triphenylphosphine oxide. In the 1H-31P decoupled NMR spectrum, the signals of the unreacted triphenylphosphine and the reacted triphenylphosphine oxide are detected at 7.4 ppm and 7.8 ppm, respectively. The ratio of the two signals is used for the calculation of the peroxide concentration. 108 oil samples with a peroxide value between 1 meq/kg and 150 meq/kg were examined using the developed method. Oils with a very low peroxide value of less than 3 meq/kg showed a relative standard deviation of 4.9%, highly oxidized oils with a peroxide value of 150 meq/kg of 0.2%. The NMR method was demonstrated as a powerful technique for the analysis of vegetable and krill oils. Another 1H NMR spectroscopic method was developed for the qualitative determination of Be2+, Sr2+ and Cd2+, and for the qualitative and quantitative determination of Ca2+, Mg2+, Hg2+, Sn2+, Pb2+ and Zn2+ by using ethylenediamine tetraacetate (EDTA) as complexing agent. EDTA is a hexadentate ligand that forms stable chelate complexes with divalent cations. The known amount of added EDTA and the signal ratio of free and complexed EDTA are used to calculate the concentrations of the divalent cations, which makes the use of an internal standard obsolete. The use of EDTA with Be2+, Sr2+, Cd2+, Ca2+, Mg2+, Hg2+, Sn2+, Pb2+ and Zn2+ result in complexes whose signals are pH-independent, showing cation-specific chemical shifts and couplings in the 1H NMR spectrum that are used for identification and quantification. In the presented NMR method, the limit of quantification of the cations Ca2+, Mg2+, Hg2+, Sn2+, Pb2+, and Zn2+ was determined with 5-22 μg/mL. This method is applicable in the food and drug sectors. The third NMR spectroscopic method introduced an alternative determination of the enantiomer excess (ee) of the chiral alcohols menthol, borneol, 1-phenylethanol and linalool using phosgene as a derivatizing reagent. Phosgene reacts with a chiral alcohol to form carboxylic acid diesters, made of two identical (RR, SS) or two different enantiomers (RS, SR). These two different types of diastereomers can be examined by the difference of their chemical shifts. In the presented method, the integration values of the carbonyl signals in the 13C NMR spectrum are used for the determination of the enantiomer excess. The limit of quantification depends, among others, on the sample and on the non-labelled or 13C-labelled phosgene used for the analysis. In the case of menthol, a quantification limit of ee=99.1% was determined using non-labelled phosgene and ee=99.9% using 13C-labelled phosgene. The 13C NMR method was also applied for the quality control of the enantiomeric purity of borneol, 1-phenylethanol and linalool. The developed 13C NMR method represents a powerful alternative to Mosher’s reagent for investigating the enantiomeric excess in chiral alcohols. This work demonstrates the variety of possibilities of applications for the quantitative nuclear magnetic resonance spectroscopy in the chemical analysis of drugs, food and chemicals using tagging reactions such as derivatizations and complexations. The nuclear resonance spectroscopic methods developed in this research work represent powerful alternatives to the previously used quality control techniques.
Salt formation is a routinely used strategy for poorly water-soluble drugs and traditionally performed with small inorganic counterions. High energy crystal lattices as well as effects on the local pH within the aqueous boundary layer during dissolution drive the increased dissolution rate and apparent solubility. Ionic liquids however, by definition low melting ionic salts with often large organic counterions, combine an increased dissolution rate with solubilization of the drug by the counterion itself. Long lasting supersaturation profiles of increased kinetic solubility were reported for several drugs formulated as ionic liquids increasing their overall bioavailability. Furthermore, aggregation and micellization between highly lipophilic compounds and amphiphilic bile acids was described before, demonstrating the capabilities of the human body itself to utilize solubilization of poorly water-soluble compounds. Development of novel counterions not only tailoring the desired physicochemical properties e.g. dissolution rate of the parent drug but adding – in a best-case scenario synergistic – pharmacological activity has been driven forward in the last years. However, salt formation can only be applied for ionizable i.e. acidic or basic compounds. While co-crystals can be used as a nonionized alternative, their formation is not always successful leading to an urgent need for other formulation strategies. In these lines, development of 2D and 3D printing techniques has been ongoing for the last decades and their pharmaceutical application has been demonstrated. The versatile nature and commercial availability allow a decentralized production further elaborating this technique for a highly flexible and patient-oriented supply with medication.
This thesis focuses on the theoretical background and potential application of salt formation in the pharmaceutical development of a drug candidate. The first section presents the current knowledge and state of the art in preparation of low melting ionic liquids i.e. salts and is translated to the in vitro investigation of molecular interaction between the poorly water-soluble drug imatinib and components of the human intestinal fluid in the second section. Development of novel antibiotic counterions and assessment of their potential use in pharmaceutical formulations with fluoroquinolones is described in the last two sections.
Chapter I describes the application of low melting ionic liquids in pharmaceutical formulation and details their development in the last two decades from versatile organic solvents in chemical synthesis towards amorphous strategies for drug delivery. The chapter gives a general overview on molecular structure and physicochemical properties of several drug containing ionic liquids and details the mechanisms which attribute to a typically fast dissolution, increased aqueous solubility as well as enhanced permeation which was reported in several publications.
Chapter II translates the increased aqueous solubility of drugs by an organic counterion to the human gastrointestinal tract with taurocholate and lecithin as main drivers for the solubilization of highly lipophilic and poorly water-soluble drugs. Investigation of the interaction of imatinib – a poorly water-soluble weak base – with fasted- and fed state simulated intestinal fluids revealed a complex interplay between the components of the intestinal fluid and the drug. Mixed vesicles and micelles were observed in concentration dependent aggregation assays and revealed differences in their size, molecular arrangement as well as composition, depending on the tested drug concentration. Overall, the study outlines the effective interaction of weakly basic drugs with taurocholate and lecithin to minimize recrystallization during intestine passage finally leading to favorable supersaturation profiles.
Chapter III focuses on the development of novel antibiotic counterions which potentially move the evolution of ionic liquids from a pharmaceutical salt with tailored physicochemical properties to a synergistic combination of two active pharmaceutical ingredients. The natural occurring anacardic acid derived from the cashew nut shell inspired a series of antibacterial active acidic compounds with increasing alkyl chain length. Their physicochemical properties, antibacterial activity, bacterial biofilm inhibition and cytotoxicity were detailed and in vivo activity in a Galleria mellonella model was assessed. This group of anacardic acid derivatives is synthetically accessible, easily modifiable and yielded two compounds with favorable activity and physicochemical profile for further drug development.
Chapter IV outlines the potential application of anacardic acid derivatives in pharmaceutical formulations by salt formation with fluoroquinolone antibiotics as well as novel techniques such as 2D/3D printing for preparation of drug imprinted products. Despite anacardic acid derivatives demonstrated promising physicochemical properties, salt formation with fluoroquinolone antibiotics was not feasible. However, 2D/3D printed samples with anacardic acid derivative alone or in combination with ciprofloxacin demonstrated physical compatibility between drug and matrix as well as antibacterial activity against three S. aureus strains in an agar diffusion assay. Conclusively, drug printing can be applied for the herein tested compounds, but further process development is necessary.
In summary, preparation of low melting ionic liquids, salts or co-crystals is an appropriate strategy to increase the aqueous solubility of poorly water-soluble drugs and tailor physicochemical properties. The counterion itself solubilizes the drug and furthermore potentially interferes with the complex micellar environment in the human intestine. However, salt formation as routinely used formulation strategy is not feasible in every case and development of alternative techniques is crucial to hurdle challenges related to unfavorable physicochemical properties. The outlined techniques for 2D/3D drug printing provide versatile production of drug products while extending the design space for novel drug development.
The charged aerosol detector (CAD) is an aerosol-based detector employed in liquid chromatography which has become established in the field of pharmaceutical analysis due to its outstanding performance characteristics, e.g. the almost uniform response for nonvolatile analytes. Owing to its principle of detection, the response of the CAD depends on the volatility of a compound and is inherently nonlinear. However, the newly implemented instrumental settings evaporation temperature and power function value (PFV) are valuable tools to overcome some of these drawbacks and can even enhance the detector’s capabilities when adjusted properly.
This thesis aimed to evaluate the impact of the new instrumental settings on the CAD performance. Additionally, the influence of modern separation techniques for small polar compounds on the CAD was assessed and the applicability of hyphenated UV-CAD techniques explored. The optimization strategies derived from the evaluation procedures and the conjunction of the instrumental and chromatographic techniques investigated were utilized for the challenging impurity profiling of amino acids and amino acid-like drugs.
The results of the method validation procedures confirmed the broad applicability of the CAD in the pharmaceutical analysis of nonvolatile compounds, supported by satisfactory sensitivity and reproducibility for meeting the regulatory requirements with respect to the ICH guidelines Q2(R1) and Q3A(R2). The limits of applicability include the analysis of semivolatile compounds, and the method transfer between current and legacy CAD models. Further advances in the definition and standardization of allowed ranges for the instrumental settings and the establishment of general optimization procedures in the method development could lead to a more widespread use of the detection technique in compendial methods.
For the quality assurance of substances for pharmaceutical use, a variety of analytical techniques are available to address specific analytical problems. In this field of application, liquid chromatography (LC) stands out as the gold standard in the pharmaceutical industry. Various detectors can be employed, which are e.g. based on UV/Vis spectroscopy for the examination of molecules with a chromophore, or mass spectrometry (MS) for structural elucidation of analytes. For the separation of enantiomers, the use of capillary electrophoresis (CE) may be more favorable due to the high separation efficiency and easy-to-use and comparatively inexpensive chiral selectors, in contrast to chiral columns for LC, which are usually very expensive and limited to a restricted number of analytes. For structure elucidation in impurity profiling, one- and multidimensional 1H NMR spectroscopy is a valuable tool as long as the analyte molecule has got nuclei that can be detected, which applies for the magnitude of organic pharmaceutical substances.
For the evaluation of the amount of mineral oil aromatic hydrocarbons (MOAH) in various paraffin samples from different suppliers, a straightforward method based on 1H NMR spectroscopy was elaborated. The MOAH/MOSH ratio was used to indicate the amount of MOAH of paraffins and to evaluate the extent of refining. In addition, a representative paraffin sample was measured without sample solvent at high temperatures (about 340 K) to avoid the interfering residual solvent signals in the spectral regions of interest. The results of both methods were in good accordance.
Moreover, the 1H NMR results were complemented with the UV measurements from the purity testing of paraffins according to the DAB 8. Correlations of the NMR and UV spectroscopic data indicated a linear relationship of both methods for the determination of MOAH in paraffins.
Finally, the 1H NMR data was evaluated by principal component analysis (PCA) to explore differences within the paraffin samples and the spectral regions in the 1H NMR spectrum which are responsible for the formation of groups. It could be found that most variation is due to the MOSH of the paraffins. The PCA model was capable of differentiating between soft, liquid and solid paraffins on the one hand and between natural and synthetic liquid paraffins on the other hand.
The impurity profiling of L-ascorbic acid 2-phosphate magnesium (A2PMg) was performed by means of one- and two-dimensional NMR spectroscopy. Several ethylated impurities could be detected, which were likely to be formed during synthesis of A2PMg. The structures of two of the ethylated impurities were identified as ascorbic acid 2-phosphate ethyl ester and ethanol, (residual solvent from synthesis). NMR spectroscopic studies of the fractions obtained from preparative HPLC of A2PMg revealed two additional impurities, which were identified as phosphorylated derivatives of ascorbic acid, ascorbic acid 3,5-phosphate and ascorbic acid 5-phosphate.
Solid state mechanochemistry as an alternative approach for stress testing was applied on the drug substances S-Ibuprofen (Ibu) and Clopidogrel (CLP) using a ball mill, in order to study their degradation profile:
First, the isomerization of S-Ibu was investigated, which was stressed in the solid state applying several milling frequencies and durations under basic, acidic and neutral conditions. For the separation of Ibu enantiomers, a chiral CE method was developed and validated according to ICH Q2(R1). It was found that S-Ibu is overall very stable to isomerization; it shows minor conversion into the R-enantiomer under basic environment applying long milling times and high frequencies.
Last, the degradation profile of clopidogrel hydrogen sulfate (CLP) was investigated, which was stressed in the solid state under various oxidative conditions. An already existing HPLC-UV method was adjusted to sufficiently separate the degradation products, which were characterized by means of UV and MS/(MS) detection. Most of the degradation products identified were already reported to result from conventional CLP stress tests. The degradation profile of CLP was mainly influenced by the material of the milling jar and the type of catalyst used.
Cell culture models are helpful tools to study inflammatory diseases, like rheumatoid arthritis (RA), osteoarthritis (OA), arteriosclerosis or asthma, which are linked to increased matrix metalloproteinase (MMP) activity. Such cell culture models often focus on the secretion of cytokines and growth factors or the direct effects of disease on tissue destruction. Even though the crucial role of MMPs in inflammatory diseases is known, the results of MMP studies are contradictious and the use of MMPs as biomarkers is inconsistent. MMPs play an important role in disease pathology, as they are involved in elastin degradation in the walls of alveoli in chronic obstructive pulmonary disease (COPD), tumor angiogenesis and metastasis and in cartilage and bone degradation in arthropathies. In RA and OA MMPs are secreted by osteocytes, synoviocytes, and by infiltrating immune cells in response to the increased concentration of inflammatory mediators, like growth factors and cytokines. MMPs are zinc and calcium-dependent proteinases and play an important role in physiological and pathological extracellular matrix (ECM) turn over. Their substrate specificity gives them the ability to degrade all major ECM components, like aggrecan, elastin, gelatin, fibronectin and all types of collagen even the triple helix of collagen monomers. The ECM consists of two large three-dimensional cross-linked macromolecule classes: one are fibrous proteins, like collagen and elastin fibers that are responsible for ECM’s structure, tensile strength, resiliency, reversible extensibility, and deformability and the second class is comprised of proteoglycans composed of glycosaminoglycan (GAG) chains covalently attached to protein cores that are multifunctionally involved in signaling pathways and cell interactions. ECM is present within all tissues and organs and changes in ECM structure contribute to pathogenesis, e.g. wounded and fibrotic tissue, COPD or tumours.
This thesis primarily focuses on the development of a diagnostic peptide system, that enables to gain information on MMP activity from ECM by deploying the isobaric mass encoding strategy. The core element of the developed system is an isotopically labelled peptide sequence (mass tag), that is released in response to elevated levels of MMPs and allows multiplexed detection in tandem mass spectrometry (LC-MS/MS). The mass reporters possess a modular structure with different functionalities. C-terminal either a transglutaminase (TG) recognition sequence or a high molecular weight polyethylene glycol (PEG) moiety was attached to immobilize the mass reporters covalently or physically at the injection site. The following matrix metalloproteinase substrate sequence (MSS) is incorporated in two different versions with different sensitivity to MMPs. The MSS were applied in pairs for relative quantification consisting of the cleavable version synthesized with natural L-amino acids and the non-cleavable D-amino acid variant. The mass tag was synthesized with isotopically labelled amino acids and is separated from the MSS by a UV light-sensitive molecule. N-terminal the mass tag is followed by a tobacco etch virus protease (TEV) sensitive sequence, that is responsible to separate the mass tag from the affinity tag, which was either the Strep-tag II sequence or biotin and were added for purification purposes.
Chapter 1 presents a step-by-step protocol on how to design a mass tag family allowing for multiplexed analysis by LC-MS/MS. The multiplexing is achieved by developing an isobar mass tag family with four family members, which are chromatographically indistinguishable, but due to the mass encoding principles they fragment in distinct y-type ions with a mass difference of 1 or 2 Da each in MS2. Furthermore, it is explained how to covalently attach the mass reporter peptides onto ECM by the activated calcium-catalyzed blood coagulation transglutaminase factor XIII (FXIIIa). The lysine of mass reporter’s TG sequence (D-domain of insulin-like growth factor-I (IGF-I)) and a glutamine in fibronectin are covalently crosslinked by FXIIIa and build an isopeptide bond. Elevated levels of MMP release the mass reporters from ECM by recognizing the inter-positioned MSS.
The designed mass reporters were able to monitor enzyme activity in an in vitro setting with cell-derived ECM, which was shown in Chapter 2. The modular structured mass reporters were investigated in a proof of concept study. First, the different modules were characterized in terms of their MMP responsiveness and their sensitivity to TEV protease and UV light. Then the FXIIIa-mediated coupling reaction was detailed and the successful coupling on ECM was visualized by an immunosorbent assay or confocal laser scanning microscopy. Finally, the immobilized mass reporters on ECM were incubated with MMP-9 to investigate their multiplexing ability of MMP activity. The cleaved mass reporter fragments were purified in three steps and mass tags were analyzed as mix of all four in LC-MS/MS.
Chapter 3 describes the change from an immobilizing system as seen in chapter 1 and 2 to a soluble enzyme activity monitoring system that was applied in an osteoarthritic mouse model. Instead of the immobilizing TG sequence the C-terminal MMS was extended with two amino acids where one holds an azide moiety to perform a strain-promoted azide-alkyne cycloaddition to a high molecular weight dibenzocyclooctyne-polyethylene glycol (DBCO-PEG), which was chosen to retain the mass reporters at the injection site. Furthermore, the N-terminal affinity tag was extended with a 2.5 kDa PEG chain to increase the half-life of the mass reporter peptides after MMP release. The systems biocompatibility was proved but its enzyme monitoring ability in an in vivo setting could not be analyzed as samples degraded during shipping resulting from the Chinese customs blocking transport to Germany.
In summary the diagnostic peptide system was developed in two variants. The immobilized version one from chapter 1 and 2 was designed to be covalently attached to ECM by the transglutaminase-mediated cross-linking reaction. In an in vitro setting the functionality of the mass reporter system for the detection of MMP activity was successfully verified. The second variant comprises of a soluble mass reporter system that was tested in an OA mouse model and showed biocompatibility. With these two designed systems this thesis provides a flexible platform based on multiplexed analysis with mass-encoded peptides to characterize cell culture models regarding their MMP activity, to deploy cell-derived ECM as endogenous depot scaffold and to develop a mass tag family that enables simultaneous detection of at least four mass tags.
Die Permeabilität von Substanzen über Biomembranen erfolgt auf Basis ihrer Größe und Lipophilie, wird jedoch auch zu einem großen Anteil vom aktiven Transport bestimmt. Speziell im menschlichen Verdauungstrakt ist dieser Transportmechanismus neben seinen essentiellen physiologischen Aufgaben, wie den Transport von Nährstoffen, an einer Resistenz gegen exogene Stoffe und Xenobiotika beteiligt, der die Aufnahme in den Organismus über einen Rücktransport in das Darmlumen limitiert. Dabei hat die membranständige Effluxpumpe p-Glykoprotein (p-GP) als ein Baustein dieses Schutzmechanismus auch einen großen Einfluss auf die Arzneimitteltherapie. Über eine Modulierung der Pharmakokinetik von Arzneistoffen beschränkt sie die Aufnahme von Medikamenten und senkt dadurch deren Bioverfügbarkeit. Es wird auch für pflanzliche Inhaltsstoffe aus der Gruppe der Polyphenole ein möglicher Einfluss auf dieses Transportprotein diskutiert. Diese Beeinflussung kann sich entweder in einer Induktion oder einer Inhibition des Proteins äußern, was positive wie negative Effekte haben kann. Eine Hemmung des Transportproteins führt zu einer erhöhten Aufnahme einiger Arzneistoffe, die mit einer erhöhten Bioverfügbarkeit und einer potentiellen Dosissenkung einhergeht. Induziert man p-GP dagegen, so wird es beispielsweise ermöglicht, potentiell schädliche Xenobiotika noch intensiver auszuscheiden und nachteilige Plasmaspiegel zu verhindern. Im Rahmen der vorliegenden Arbeit sollte daher der Einfluss ausgewählter Polyphenole auf die Funktionalität und die Genexpression im CaCo-II-Zellkulturmodell näher untersucht, sowie vorab charakteristische Eigenschaften der pflanzlichen Inhaltsstoffe - Taxifolin, Silibinin, M1, Urolithin A, Urolithin B, Urolithin C, Isourolithin A, racemisches Hydnocarpin D, (+)-Hydnocarpin D, (-)-Hydnocarpin D - vergleichend bestimmt werden. Diese stoffspezifischen Charakteristika umfassten die Zytotoxizität, die Stabilität und die antioxidative Kapazität. Vor allem die Zytotoxizität und die Stabilität sind essentielle Parameter für aussagekräftige Resultate. Die Substanzen waren in der eingesetzten Konzentration von 50 µM mehrheitlich, mit Ausnahme des Hydnocarpins D, nicht-toxisch innerhalb der relevanten Versuchszeiträume, 4 h und 24 h, und den verwendeten Kulturmedien, DMEM-Pest und HBSS. Vor allem im Hinblick auf die Genexpressionsversuche war es die Basis für valide Ergebnisse, den Zeitraum bis 24 h als nicht-toxisch sicherstellen zu können. Hinsichtlich der Stabilität waren nur Taxifolin (27 % Restkonzentration) und der M1 (0 % Restkonzentration) nach 24 h in Zellkulturmedium kritisch. Auf Basis ihrer antioxidativen Kapazität werden pflanzlichen Inhaltsstoffen eine Reihe von gesundheitsförderlichen Merkmalen nachgesagt, weswegen dieser Aspekt für die Testsubstanzen zusätzlich vergleichend evaluiert wurde. Der Eintritt von Pathogenen kann Zusammenfassung 377 zum Beispiel durch oxidative Schädigung des Darmepithels erleichtert werden, was zusätzlich zu einem Effekt auf p-GP durch die Polyphenole unter Umständen positiv beeinflusst werden kann. Taxifolin, der M1 sowie die Urolithine A und C konnten so als antioxidativ aktive Stoffe erstmals vergleichend analysiert und die Resultate sinnvoll zu bestehenden Daten in Relation gesetzt werden. Sie konnten nach antioxidativer Potenz in der Reihenfolge Urolithin C > M1 > Taxifolin > Urolithin A geordnet werden. Zur Analyse des Einflusses der ausgewählten Polyphenole auf die Funktionalität von p-GP sollten Transportversuche über einen CaCo-II-Monolayer mit Rhodamin 123 als Markersubstanz durchgeführt werden. Diese Untersuchungen benötigen typischerweise eine vorbereitende Kulturzeit der Zellen von insgesamt drei Wochen, sodass sich eine Verkürzung dieser Zeitspanne aus Zeitersparnis- und Kostengründen positiv auf den Durchsatz der Versuche auswirken würde. In einem umfassenden Ansatz mit kombinierter Bestimmung der Qualifizierung der Zellschichten im Hinblick auf Qualität des Monolayers (TEER-Messung, Lucifer-Yellow-Transportrate, Fluoreszenzfärbung der Tight-junctions) sowie der Funktionalität und Expression von p-GP gelang der Nachweis, dass 14 Tage hinreichend und sinnvoll waren. Zentraler Bestandteil war in der vorliegenden Arbeit die Identifizierung der Effekte der Urolithine auf sowohl p-GP direkt, als auch auf die Genexpression dieses Transportproteins. Diese Polyphenole werden im menschlichen Verdauungstrakt über einen bakteriellen Metabolismus aus Ellagtanninen und Ellagsäure hergestellt und sind aufgrund ihrer vielfältigen gesundheitsförderlichen Charakteristiken in der Forschung von steigendem Interesse. Hierfür konnten nach unserem Kenntnisstand mit den gewählten Versuchsansätzen neue Erkenntnisse gewonnen werden. In den Transportversuchen mit Rhodamin 123 als Modellsubstrat von p-GP konnten die Urolithine den p-GP-vermittelten Transport positiv beeinflussen. Die Urolithine B (Papp-Ratio 1,98), C (Papp-Ratio 2,15) und das Isourolithin A (Papp-Ratio 1,63) steigerten den Rhodamintransport signifikant und lediglich für Urolithin A (Papp-Ratio 1,45) konnte keine Signifikanz belegt werden. Der Einfluss der Urolithine lag jeweils im Bereich des Modellinduktors Dexamethason. Ebenso konnte eine positive Modulierung der Genexpression nach 24 h detektiert werden. Die Hochregulierungen durch die Urolithine A (zwei- bis dreifach), B (1,4-fach) und C (1,8-fach) waren konsistent und statistisch signifikant. Urolithin A konnte hierbei als potentester Induktor charakterisiert werden, wohingegen sein Isomer Isourolithin A keinerlei signifikante Beeinflussung der Expression zeigte. In diesen Inkubationsversuchen wurde die Eigenschaft zur Erhöhung der Genexpression über den Einfluss auf den p-GP-vermittelten Rhodamintransport bestätigt. Die Urolithine A, B, C und Isourolithin A konnten nach einer Vorinkubation über 24 h und 48 h auch den Transport von Rhodamin 123 nochmals signifikanter zu den klassischen E Zusammenfassung 378 Transportversuchen ohne Vorinkubation steigern. Relevanz hierfür hatte der erste Zeitraum über 24 h, da hier ein deutlicher Anstieg der Rhodamintransportrate zu erkennen war. Nach 48 h stieg der Rhodamintransport nur noch geringfügig an oder ging sogar leicht zurück (Urolithin B). Hinsichtlich der Genexpression konnte nach 48 h nur noch Urolithin C p-GP signifikant hochregulieren, allerdings sind diese Erkenntnisse auf Basis der Zytotoxizität der Substanzen über diesen Zeitraum kritisch zu betrachten. In der Analyse des Effektes der weiteren Polyphenole auf die Genexpression von p-GP konnten für die meisten Stoffe nur zufällige Zusammenhänge hinsichtlich Hoch- und Herunterregulierung bestimmt werden. In den Transportversuchen konnte jedoch (+)-Hydnocarpin (Papp-Ratio 0,48) den Transport in gleichem Ausmaß wie der Modellinhibitor Verapamil (Papp-Ratio 0,48) hemmen. Durch Modifizierung des Versuchsmediums zur Annäherung an physiologischeren Bedingungen (Gallensäuren, pH 6) konnte für manche Substanzen ein deutlich verändertes Verhalten beobachtet werden. Die Rhodamintransportrate nahm unter Einfluss von Urolithin B, Isourolithin A und dem M1 signifikant nun ab und bei Urolithin C signifikant zu. Dies legt nahe, dass mit dem klassischen Transportversuchsmodell lediglich Tendenzen für die Substanzen bestimmt werden können. Weitere Untersuchungen näher an der Physiologie des Verdauungstraktes sind nötig, um ein genaueres Bild des Stoffeinflusses zu gewinnen. Die Frage nach zeitlichem Einsetzen beziehungsweise der Kontinuität des Effektes auf p� GP konnte mit den Urolithinen A, B und C sowie Dexamethason geklärt werden. Eine Substanzexposition von lediglich fünf Minuten war nicht ausreichend, um in den nachfolgenden zwei Stunden einen Effekt zu beobachten. Dies legt eine Reversibilität der zugrundeliegenden Mechanismen und eine notwendige dauerhafte Anwesenheit der Substanzen über die Versuchszeit nahe. Neben Rhodamin 123 wurden noch Transportversuche mit dem Fluorchinolonantibiotikum Ciprofloxacin als Modellsubstanz durchgeführt, da es aufgrund dessen Substratcharakters für p-GP von therapeutischer Relevanz sein kann, wenn das Transportverhalten durch Polyphenole beeinflusst wird. Im Gegensatz zu Rhodamin 123 wurde der Transport von Ciprofloxacin durch die vier Urolithine verringert, was für diese Metabolismusprodukte eine zusätzliche Wirkung auf weitere Transportproteine nahelegt, weil Ciprofloxacin unter anderem auch über BRCP transportiert wird. Mittels des bakteriellen Endotoxins LPS konnte eine Schädigung des CaCo-II-Monolayers erzeugt werden, welche sich über erniedrigte TEER-Werte und einen erhöhten Rhodamintransport nachweisen ließ. Eine Vorinkubation der vier Urolithine war nicht in der Lage, diese Schädigung abzumildern, jedoch nicht komplett zu verhindern. Die TEER- Zusammenfassung 379 Werte konnten zwar wieder etwas gesteigert werden, jedoch maskierte die starke Stimulation dieser Pflanzenstoffe auf p-GP und den damit verbundenen Transport von Rhodamin 123 mögliche positive Effekte auf diese oxidative Stresssituation. Zusammenfassend war es mit der vorliegenden Arbeit erstmals durch systematische vergleichende Untersuchung und Kombination von Charakterisierungsansätzen möglich, eine deutliche Beeinflussung der Genexpression und Funktionalität des p-Glykoproteins durch vor allem die Urolithine aufzuzeigen, was eine Relevanz sowohl des Mikrobioms als auch der Ernährung in der Arzneimitteltherapie nahelegt. Zudem gelang es den klassischen Transportassay durch Verkürzung um eine Woche zu verbessern.
The GABA\(_{B}\) receptor agonist baclofen is a medication commonly used for the treatment of muscle spasticity. It is an amino acid and related to the neurotransmitter GABA. In this study, we developed a new, gradient high-performance liquid chromatography (HPLC) method for the impurity assessment of baclofen, which is appropriate for pharmacopoeial purposes. Since the impurities related to the synthesis pathway are acids, zwitterionic, or neutral, the method development is challenging. However, the separation of all components was achieved on a C18 stationary phase using a water–acetonitrile–trifluoroacetic acid gradient. A limit of detection (LOD) of at least 0.02% was registered for all specified impurities. Additionally, CAD detection was performed to detect potential impurities lacking off a chromophore. The baclofen batches analyzed are far more pure than expected. All impurities were found below the specification limit, and thus, they can be regarded as unspecified. Moreover, the required runtime could be significantly reduced compared to the current USP or Ph. Eur. method.
Inhibition of coronavirus (CoV)‐encoded papain‐like cysteine proteases (PL\(^{pro}\)) represents an attractive strategy to treat infections by these important human pathogens. Herein we report on structure‐activity relationships (SAR) of the noncovalent active‐site directed inhibitor (R)‐5‐amino‐2‐methyl‐N‐(1‐(naphthalen‐1‐yl)ethyl) benzamide (2 b), which is known to bind into the S3 and S4 pockets of the SARS‐CoV PL\(^{pro}\). Moreover, we report the discovery of isoindolines as a new class of potent PL\(^{pro}\) inhibitors. The studies also provide a deeper understanding of the binding modes of this inhibitor class. Importantly, the inhibitors were also confirmed to inhibit SARS‐CoV‐2 replication in cell culture suggesting that, due to the high structural similarities of the target proteases, inhibitors identified against SARS‐CoV PL\(^{pro}\) are valuable starting points for the development of new pan‐coronaviral inhibitors.