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All presented studies aimed on the improvement of the quality analysis of already monographed drugs. Thereby different LC methods were applied and coupled to i.e., the UV/VIS detector, the CAD or a hyphenation of these detectors, respectively. The choice of the chromatographic system including the detector was largely dependent on the physicochemical properties of the respective analytes.
With the risk-assessment report on the API cetirizine we presented an exemplary tool, that can help to minimize the risk of the occurrence of unexpected impurities. An in- deep analysis of each step within synthesis pathway by means of reaction matrices of all compounds was performed. It is essential to understand the complete impurity profile of all reactants, solvents, and catalysts and to include them in the matrix. Finally, the API of this synthesis was checked if all impurities are identified by this tool. Of note, a shortcoming of such a targeted approach is that impurities can still occur, but they are not captured. This disadvantage can be partially compensated by non-targeted approaches if they are performed in parallel with the other studies that represent most of the impurities. However, this work also shows that even in a supposedly simple synthesis, potentially hundreds of by-products can be formed. For each of them, it must be decided individually whether their formation is probable or how their quantity can be minimized in order to obtain APIs, that are as pure as possible.
In the dapsone project it was aimed to replace the existing old Ph. Eur. TLC method with a modern RP-HPLC method. This was successful and since Ph. Eur. 10.6, the method developed in this work, became a valid monograph. Within the revision process of the monograph, the individual limits for impurities were tightened. However, this new method needs HPLC instrumentation, suitable to perform gradients. As this is not always available in all control laboratories, we also developed an alternative, more simple method using two different isocratic runs for the impurity analysis. The obtained batch results of both, the new pharmacopoeial method and the more simple one, were in a comparable order of magnitude. Furthermore, within the method development stage of the Ph. Eur. method, we could identify one unknown impurity of the impurity reference by high-resolution MS/MS analysis.
Also, in the baclofen project it was aimed to replace the existing Ph. Eur. method with the introduction of an additional impurity to be quantified. A corresponding method was developed and validated. However, due to the harmonization process of the pharmacopoeias, it is currently not used. In addition, we tried to find further, non- 116
SUMMARY
chromophoric impurities by means of the CAD. However, except for one counterion of an impurity, no further impurities were found. Also, the aforementioned new impurity could not be detected above the reporting threshold in the batches analyzed. As the only individually specified impurity A is also present at a low level, it can be concluded that the examined batches of baclofen are very pure.
The use of universal detectors, such as the CAD can be particularly interesting for compounds with no chromophore or those with only a weak chromophore. Therefore, we decided to take a closer look at the impurity profile of acarbose. Currently, acarbose and its impurities are being studied by low wavelength UV detection at 210 nm. Therefore, the question arose whether there are no other impurities in the API that do not show absorption at this wavelength. CAD, which offers consistent detection properties for all non-volatile compounds, is ideally suited for this purpose. However, it was not so easy to use the CAD together with the UV detector, for example, as a hyphenated detection technique, because the Ph. Eur. method uses phosphate buffers. However, this is non-volatile and therefore inappropriate for the CAD. Therefore, an attempt was made to replace the buffer with a volatile one. However, since this did not lead to satisfactory results and rather the self-degradation process of the stationary phase used could be observed by means of the CAD, it was decided to switch to alternative stationary phases. A column screening also revealed further difficulties with acarbose and its impurities: they show an epimerization reaction at the end of the sugar chain. However, since one wanted to have uniform peaks in the corresponding chromatograms, one had to accelerate this reaction significantly to obtain only one peak for each component. This was best achieved by using two stationary phases: PGC and Amide-HILIC. Impurity-profiling methods could be developed on each of the two phases. In addition, as expected, new impurities could be detected, albeit at a low level. Two of them could even be identified by spiking experiments as the sugar fragments maltose and maltotriose.
Taken together, it can be concluded, that this work has contributed significantly to the improvement of the quality analysis of monographed drugs. In addition to the presented general tool for the identification of potential impurities, one of the methods developed, had already been implemented to the Ph. Eur. In an effort to improve the CAD's universal detection capabilities, additional methods have also been developed. Further, new improved methods for the impurity profiling are ready to use.
Formation of the Aurora-A–MYCN complex increases levels of the oncogenic transcription factor MYCN in neuroblastoma cells by abrogating its degradation through the ubiquitin proteasome system. While some small-molecule inhibitors of Aurora-A were shown to destabilize MYCN, clinical trials have not been satisfactory to date. MYCN itself is considered to be `undruggable' due to its large intrinsically disordered regions. Targeting the Aurora-A–MYCN complex rather than Aurora-A or MYCN alone will open new possibilities for drug development and screening campaigns. To overcome the challenges that a ternary system composed of Aurora-A, MYCN and a small molecule entails, a covalently cross-linked construct of the Aurora-A–MYCN complex was designed, expressed and characterized, thus enabling screening and design campaigns to identify selective binders.
Amber Light Control of Peptide Secondary Structure by a Perfluoroaromatic Azobenzene Photoswitch
(2023)
The incorporation of photoswitches into the molecular structure of peptides and proteins enables their dynamic photocontrol in complex biological systems. Here, a perfluorinated azobenzene derivative triggered by amber light was site‐specifically conjugated to cysteines in a helical peptide by perfluoroarylation chemistry. In response to the photoisomerization (trans→cis) of the conjugated azobenzene with amber light, the secondary structure of the peptide was modulated from a disorganized into an amphiphilic helical structure.
Dietary fatty acids serve as objective biomarkers for the estimation of habitual diet mainly because biomarkers are free of memory bias or inaccuracies of food databases. The aim of the present work encompassed the implementation of a gas chromatographical method coupled with a mass spectrometrical and flame-ionization detector for analysis of fatty acid biomarkers in human biospecimens, their analytical determination and statistical evaluation in two different study populations and different biospecimens as well as the elaboration of adverse reactions to food ingredients with special focus on food allergies and food intolerances in the context of a possible implementation into an application for consumer health. The first aim was the identification of potential influence of fatty acid biomarkers on desaturase and elongase indexes (Δ9DI, Δ6DI, Δ5DI and ELOVLI5), which are factors in type 2 diabetes risk, in breast adipose tissue from healthy women. Influence of further variables on respective indexes was also investigated. 40 samples were investigated and potential variables were either collected by questionnaire or determined. Principle component analysis was applied for fatty acid biomarkers (PCdiet1, PCdiet2 and PCdiet3 representative for the dietary intake of vegetable oils/nuts, fish and partially hydrogenated vegetable oils), endogenous estrogens (PCE1) and oxysterols (PCOxy1). Multiple linear regression models were applied. Δ9DI and Δ6DI were influenced non-significantly and significantly negatively by PCdiet2 supporting a putative beneficial effect of vegetable oils and nuts on type 2 diabetes risk factors. ELOVLI5 and Δ5DI were influenced significantly and non-significantly positively by PCdiet1 supporting a putative beneficial effect of fish consumption on type 2 diabetes risk factors. On the other hand, PCdiet1 also significantly and non-significantly positively influenced Δ9DI and Δ6DI supporting a putative adverse effect of fish biomarkers on type 2 diabetes risk factors. The opposing influences of PCdiet1 suggesting an ambivalent role of dietary intake of fish on investigated indexes. Δ6DI was significantly positively influenced by PCdiet3 and number of pregnancies supporting a putative adverse effect of partially hydrogenated vegetable oils and pregnancies on type 2 diabetes risk factors. Lifestyle factors like smoking significantly and non-significantly influenced Δ9DI and Δ6DI putatively adversely. Δ5DI was influenced significantly positively by estrogen active drugs suggesting a putative beneficial effect on type 2 diabetes risk factors. It must be considered that a variation coefficient of up to 0.44 only explained 44% of variance of the respective indexes, suggesting other influencing factors might play a role. The second aim was the implementation of a gas chromatographical method coupled with a mass spectrometrical and flame-ionization detector for analysis of fatty acid biomarkers in human biospecimens. The method was optimized for separation and detection of 40 fatty acids. Mean recovery for tridecanoic acid was x(tridecanoic acid) = 90.51% and for nonadecanoic acid x(nonadecanoic acid) = 96.21%. Thus, there was no significant loss of fatty acids with shorter and longer carbon chains over the extraction process to be expected. Limit of detections were calculated in adipose tissue samples and ranged from 0.007 to 0.077% of the proportion of the respective fatty acid to total fatty acids. The third aim was the investigation if differentiation between breast glandular and adipose tissue had a relevant impact on the analysis of dietary fatty acid biomarkers or if contamination of breast glandular with breast adipose tissue and vice versa was neglectable for the analysis of dietary fatty acid biomarkers. No statistical significant differences were observed for all investigated fatty acid biomarkers (pentadecanoic-, heptadecanoic-, trans palmitoleic-, eicosapentaenoic-, docosahexaenoic-, linoleic and α-linolenic acid) between breast glandular and adipose tissue. Thus, differentiation between breast glandular and adipose tissue seems not to be necessary for the analysis of fatty acids serving as biomarkers for the intake of specific food groups. Potential influence of mixed breast tissue on fatty acid biomarkers analysis seems to be neglectable. The fourth aim was the determination of fatty acid biomarkers in adipose tissue in another study population from healthy participants. 27 adipose tissue samples were analyzed. Milk and ruminant fat biomarkers exhibited proportions of 0.47% for pentadecanoic acid, 0.34% for heptadecanoic acid and 0.25% for trans palmitoleic acid. Fish fatty acid biomarkers revealed proportions of 0.034% for eicosapentaenoic acid and 0.061% for docosahexaenoic acid. The mean proportion of vegetable oils and nuts biomarkers were 9.58% for linoleic acid and 0.48% for α-linolenic acid in all adipose tissues. Principle component analysis was applied for the fatty acid biomarkers to provide objective markers of habitual diet for this study population. PCdiet1 was mainly characterized by pentadecanoic acid, heptadecanoic acid and trans palmitoleic acid and therefore served as a principle component for the dietary intake of milk and ruminant fat. PCdiet2 and PCdiet3 only exhibited pattern for ω3 and ω6 fatty acids but not for dietary intake of specific food groups and could therefore not used as objective marker. PCdiet1, 2 and 3 explained 82.76% of variance. The last aim of this thesis was the elaboration of adverse reactions to food ingredients with special focus on food allergies and food intolerances in the context of a possible implementation into an application for consumer health. Scientific information on adverse reactions to food ingredients and trigger substances was provided in this thesis and possible implementation strategies were evaluated. For food allergens, which have regulatory requirements in the context of labelling, a strategy was elaborated, where it is necessary to provide information on the list of ingredients, the nexus ’contain’ and the respective food allergen as well as information on the name of the product. For food intolerances, which do not have regulatory requirements, limits were shown in the context of the application. If the elaborated food intolerances shall be implemented into the application, a professional dietary concept has to be developed for every food intolerance because of the complexity of the implementation.
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.
Azobenzene derivatives with activity against drug‐resistant Candida albicans and Candida auris
(2023)
Increasing resistance against antimycotic drugs challenges anti‐infective therapies today and contributes to the mortality of infections by drug‐resistant Candida species and strains. Therefore, novel antifungal agents are needed. A promising approach in developing new drugs is using naturally occurring molecules as lead structures. In this work, 4,4'‐dihydroxyazobenzene, a compound structurally related to antifungal stilbene derivatives and present in Agaricus xanthodermus (yellow stainer), served as a starting point for the synthesis of five azobenzene derivatives. These compounds prevented the growth of both fluconazole‐susceptible and fluconazole‐resistant Candida albicans and Candida auris strains. Further in vivo studies are required to confirm the potential therapeutic value of these compounds.
The aim of this study was to determine the potential of some Ghanaian underutilized legumes in helping to reduce the problems of poverty, hunger and malnutrition among the vulnerable group of the Ghanaian population. The study looked into the functional properties, fat and fatty acid distribution, raffinose, sucrose, glucose, fructose, calcium, magnesium, sodium, potassium, iron, copper, manganese, zinc, cyanide and isoflavone contents of raw and processed seed flours of Cajanus cajan, Canavalia ensiformis, Canavalia gladiata, Mucuna pruriens, Parkia biglobosa, Phaseolus lunatus and Vigna subterranea. The parameters mentioned above were also determined for raw fruit flour of Dialium guineense. In addition to these, the study also looked into the crude protein and starch contents of the raw and processed seed flours of Canavalia gladiata, Parkia biglobosa and Vigna subterranea. The obtained results suggest that the legumes may have untapped potential, which may be exploited to help assist in reducing hunger, malnutrition and poverty in Ghana. Results of the functional properties reveal that the legumes may serve useful roles in various food products. For instance, velvet tamarind (Dialium guineense) flour may be useful in infant food formulations because of it high solubility and low bulk density. African Locust bean (Parkia biglobosa) flour had the highest fat content among the studied flours, recording a fat content of approximately 14%. It may therefore be economical to express the oil and use the oil as an edible oil or for industrial applications for products such as soaps, shampoos, paints, etc. This means the properties of the oil of African Locust bean flour need to be studied to know the uses of the oil. Unsaturated fatty acids in the cis configuration formed more than 50% of the fatty acids in all the legumes. This observation coupled with the low sodium content of all the legumes suggest that these legumes may be suitable for consumption to prevent cardiovascular diseases. The daily nutrient needs of individuals can be met by the consumption of the appropriate amounts of these legumes. For example, 375.25 g of processed velvet beans (Mucuna pruriens) flour may be able to meet the adequate intake (AI) of 350 mg/day magnesium for adult males.
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.
Oral antineoplastic drugs are an important component in the treatment of solid tumour diseases, haematological and immunological malignancies. Oral drug administration is associated with positive features (e.g., non-invasive drug administration, outpatient care with a high level of independence for the patient and reduced costs for the health care system). The systemic exposure after oral intake however is prone to high IIV as it strongly depends on gastrointestinal absorption processes, which are per se characterized by high inter-and intraindividual variability. Disease and patient-specific characteristics (e.g., disease state, concomitant diseases, concomitant medication, patient demographics) may additionally contribute to variability in plasma concentrations between individual patients. In addition, many oral antineoplastic drugs show complex PK, which has not yet been fully investigated and elucidated for all substances. All this may increase the risk of suboptimal plasma exposure (either subtherapeutic or toxic), which may ultimately jeopardise the success of therapy, either through a loss of efficacy or through increased, intolerable adverse drug reactions. TDM can be used to detect suboptimal plasma levels and prevent permanent under- or overexposure. It is essential in the treatment of ACC with mitotane, a substance with unfavourable PK and high IIV. In the current work a HPLC-UV method for the TDM of mitotane using VAMS was developed. A low sample volume (20 µl) of capillary blood was used in the developed method, which facilitates dense sampling e.g., at treatment initiation. However, no reference ranges for measurements from capillary blood are established so far and a simple conversion from capillary concentrations to plasma concentrations was not possible. To date the therapeutic range is established only for plasma concentrations and observed capillary concentrations could not be reliable interpretated.The multi-kinase inhibitor cabozantinib is also used for the treatment of ACC. However, not all PK properties, like the characteristic second peak in the cabozantinib concentration-time profile have been fully understood so far. To gain a mechanistic understanding of the compound, a PBPK model was developed and various theories for modelling the second peak were explored, revealing that EHC of the compound is most plausible. Cabozantinib is mainly metabolized via CYP3A4 and susceptible to DDI with e.g., CYP3A4 inducers. The DDI between cabozantinib and rifampin was investigated with the developed PBPK model and revealed a reduced cabozantinib exposure (AUC) by 77%. Hence, the combination of cabozantinib with strong CYP inducers should be avoided. If this is not possible, co administration should be monitored using TDM. The model was also used to simulate cabozantinib plasma concentrations at different stages of liver injury. This showed a 64% and 50% increase in total exposure for mild and moderate liver injury, respectively.Ruxolitinib is used, among others, for patients with acute and chronic GvHD. These patients often also receive posaconazole for invasive fungal prophylaxis leading to CYP3A4 mediated DDI between both substances. Different dosing recommendations from the FDA and EMA on the use of ruxolitinib in combination with posaconazole complicate clinical use. To simulate the effect of this relevant DDI, two separate PBPK models for ruxolitinib and posaconazole were developed and combined. Predicted ruxolitinib exposure was compared to observed plasma concentrations obtained in GvHD patients. The model simulations showed that the observed ruxolitinib concentrations in these patients were generally higher than the simulated concentrations in healthy individuals, with standard dosing present in both scenarios. According to the developed model, EMA recommended RUX dose reduction seems to be plausible as due to the complexity of the disease and intake of extensive co-medication, RUX plasma concentration can be higher than expected.
Characterization of binding properties of ephedrine derivatives to human alpha-1-acid glycoprotein
(2023)
Most drugs, especially those with acidic or neutral moieties, are bound to the plasma protein albumin, whereas basic drugs are preferentially bound to human alpha-1-acid glycoprotein (AGP). The protein binding of the long-established drugs ephedrine and pseudoephedrine, which are used in the treatment of hypotension and colds, has so far only been studied with albumin. Since in a previous study a stereoselective binding of ephedrine and pseudoephedrine to serum but not to albumin was observed, the aim of this study was to check whether the enantioselective binding behavior of ephedrine and pseudoephedrine, in addition to the derivatives methylephedrine and norephedrine, is due to AGP and to investigate the influence of their different substituents and steric arrangement. Discontinuous ultrafiltration was used for the determination of protein binding. Characterization of ligand-protein interactions of the drugs was obtained by saturation transfer difference nuclear magnetic resonance spectroscopy. Docking experiments were performed to analyze possible ligand-protein interactions. The more basic the ephedrine derivative is, the higher is the affinity to AGP. There was no significant difference in the binding properties between the individual enantiomers and the diastereomers of ephedrine and pseudoephedrine.