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Insulin-like growth factor-I (IGF-I) is a 70-amino acid polypeptide with a molecular weight of approximately 7.6 kDa acting as an anabolic effector. It is essential for tissue growth and remodeling. Clinically, it is used for the treatment of growth disorders and has been proposed for various other applications including musculoskeletal diseases. Unlike insulin, IGF-I is complexed to at least six high-affinity binding proteins (IGFBPs) exerting homeostatic effects by modulating IGF-I availability to its receptor (IGF-IR) on most cells in the body as well as changing the distribution of the growth factor within the organism.1-3 Short half-lived IGF-I have been the driving forces for the design of localized IGF-I depot systems or protein modification with enhanced pharmacokinetic properties. In this thesis, we endeavor to present a versatile biologic into which galenical properties were engineered through chemical synthesis, e.g., by site-specific coupling of biomaterials or complex composites to IGF-I. For that, we redesigned the therapeutic via genetic codon expansion resulting in an alkyne introduced IGF-I, thereby becoming a substrate for biorthogonal click chemistries yielding a site-specific decoration.
In this approach, an orthogonal pyrrolysine tRNA synthetase (PylRS)/tRNAPyl CUA pair was employed to direct the co-translational incorporation of an unnatural amino acid—¬propargyl-L-lysine (plk)—bearing a clickable alkyne functional handle into IGF-I in response to the amber stop codon (UAG) introduced into the defined position in the gene of interest. We summarized the systematic optimization of upstream and downstream process alike with the ultimate goal to increase the yield of plk modified IGF-I therapeutic, from the construction of gene fusions resulting in (i) Trx-plk-IGF-I fusion variants, (ii) naturally occurring pro-IGF-I protein (IGF-I + Ea peptide) (plk-IGF-I Ea), over the subsequent bacterial cultivation and protein extraction to the final chromatographic purification. The opportunities and hurdles of all of the above strategies were discussed. Evidence was provided that the wild-type IGF-I yields were pure by exploiting the advantages of the pHisTrx expression vector system in concert with a thrombin enzyme with its highly specific proteolytic digestion site and multiple-chromatography steps. The alkyne functionality was successfully introduced into IGF-I by amber codon suppression. The proper folding of plk-IGF-I Ea was assessed by WST-1 proliferation assay and the detection of phosphorylated AKT in MG-63 cell lysate. The purity of plk-IGF-I Ea was monitored with RP-HPLC and SDS-PAGE analysis. This work also showed site-specific coupling an alkyne in plk-IGF-I Ea by copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) with potent activities in vitro. The site-specific immobilization of plk-IGF-I Ea to the model carrier (i.e., agarose beads) resulted in enhanced cell proliferation and adhesion surrounding the IGF-I-presenting particles. Cell proliferation and differentiation were enhanced in the accessibility of IGF-I decorated beads, reflecting the multivalence on cellular performance.
Next, we aimed at effectively showing the disease environment by co-delivery of fibroblast growth factor 2 (FGF2) and IGF-I, deploying localized matrix metalloproteinases (MMPs) upregulation as a surrogate marker driving the response of the drug delivery system. For this purpose, we genetically engineered FGF2 variant containing an (S)-2-amino-6-(((2-azidoethoxy)carbonyl)amino)hexanoic acid incorporated at its N-terminus, followed by an MMPs-cleavable linker (PCL) and FGF2 sequence, thereby allowing site-directed, specific decoration of the resultant azide-PCL-FGF2 with the previously mentioned plk-IGF-I Ea to generate defined protein-protein conjugates with a PCL in between. The click reaction between plk-IGF-I Ea and azide-PCL-FGF2 was systematically optimized to increase the yield of IGF-FGF conjugates, including reaction temperature, incubation duration, the addition of anionic detergent, and different ratios of the participating biopharmaceutics. The challenge here was that CuAAC reaction components or conditions might oxidize free cysteines of azide-PCL-FGF2 and future work needs to present the extent of activity retention after conjugation. Furthermore, our study provides potential options for dual-labeling of IGF-I either by the introduction of unnatural amino acids within two distinct positions of the protein of interest for parallel “double-click” labeling of the resultant plk-IGF-I Ea-plk or by using a combination of enzymatic-catalyzed and CuAAC bioorthogonal coupling strategies for sequentially dual-labeling of plk-IGF-I Ea.
In conclusion, genetic code expansion in combination with click-chemistry provides the fundament for novel IGF-I analogs allowing unprecedented site specificity for decoration. Considerable progress towards IGF-I based therapies with enhanced pharmacological properties was made by demonstrating the feasibility of the expression of plk incorporated IGF-I using E. coli and retained activity of unconjugated and conjugated IGF-I variant. Dual-labeling of IGF-I provides further insights into the functional requirements of IGF-I. Still, further investigation warrants to develop precise IGF-I therapy through unmatched temporal and spatial regulation of the pleiotropic IGF-I.
Articular cartilage lesions that occur upon intensive sport, trauma or degenerative disease represent a severe therapeutic problem. At present, osteoarthritis is the most common joint disease worldwide, affecting around 10% of men and 18% of women over 60 years of age (302). The poor self-regeneration capacity of cartilage and the lack of efficient therapeutic treatment options to regenerate durable articular cartilage tissue, provide the rationale for the development of new treatment options based on cartilage tissue engineering approaches (281). The integrated use of cells, biomaterials and growth factors to guide tissue development has the potential to provide functional substitutes of lost or damaged tissues (2,3). For the regeneration of cartilage, the availability of mesenchymal stromal cells (MSCs) or their recruitment into the defect site is fundamental (281). Due to their high proliferation capacity, the possibility to differentiate into chondrocytes and their potential to attract other progenitor cells into the defect site, bone marrow-derived mesenchymal stromal cells (BMSCs) are still regarded as an attractive cell source for cartilage tissue engineering (80). However, in order to successfully engineer cartilage tissue, a better understanding of basic principles of developmental processes and microenvironmental cues that guide chondrogenesis is required.
Quinolone antibiotics present an attractive oral treatment option in patients with cystic fibrosis (CF). Prior studies have reported comparable clearances and volumes of distribution in patients with CF and healthy volunteers for primarily renally cleared quinolones. We aimed to provide the first pharmacokinetic comparison for pefloxacin as a predominantly nonrenally cleared quinolone and its two metabolites between both subject groups. Eight patients with CF (fat-free mass [FFM]: 36.3 ± 6.9 kg, average ± SD) and ten healthy volunteers (FFM: 51.7 ± 9.9 kg) received 400 mg pefloxacin as a 30 min intravenous infusion and orally in a randomized, two-way crossover study. All plasma and urine data were simultaneously modelled. Bioavailability was complete in both subject groups. Pefloxacin excretion into urine was approximately 74% higher in patients with CF compared to that in healthy volunteers, whereas the urinary excretion of metabolites was only slightly higher in patients with CF. After accounting for body size and composition via allometric scaling by FFM, pharmacokinetic parameter estimates in patients with CF divided by those in healthy volunteers were 0.912 for total clearance, 0.861 for nonrenal clearance, 1.53 for renal clearance, and 0.916 for volume of distribution. Nonrenal clearance accounted for approximately 90% of total pefloxacin clearance. Overall, bioavailability and disposition were comparable between both subject groups.
Development of Novel Quinolone Amides Against the African Sleeping Sickness - A Fluorine Walk
(2019)
In recent years the transmission of the Human African Trypanosomiasis could be significantly reduced. The reported cases in 2016 reached a historic low level of 2184 cases and these achievements can be ascribed to intense control and surveillance programmes.118 However, most of the reported cases (>1000 in 2015) occurred in the Democratic Republic of the Congo and thus, need to be treated adequately. In particular, when the parasites have traversed the blood-brain barrier (BBB), treatment proved to be even more difficult. In addition, the number of cases always came in waves due to many reasons, e.g., development of resistances. Thus, it can be expected from experiences of the past that the number of cases will increase again. Hence, novel chemical entities are desperately needed in order to overcome the drawbacks which are associated with the current treatment options.
Our drug discovery approach included an initial drug repurposing strategy combined with a phenotypic screening. S. Niedermeier found novel active compounds derived from commercial fluoroquinolones. The most promising hit compound was further developed by G. Hiltensperger resulting in the lead quinolone amide GHQ168 (IC50 = 0.047 µM).
This doctoral thesis is about new insights into the SAR of the quinolone amides and the enhancement of the lead compound. Special consideration was given to the fluorine atom in the quinolone amides and how certain fluorine substitution patterns influence the antitrypanosomal activity, physicochemical properties and pharmacokinetics (i.e. ‘fluorine walk’). Moreover, the ability of the compound class crossing the BBB should be investigated. This feature is inevitable necessary in order to potentially treat African sleeping sickness stage II.
The Gould-Jacobs protocol was predominantly used for the synthesis of the quinolone core. Since former SAR studies mainly concentrated on the variation in positions 1, 3 and 7, quinolone scaffolds (2a-i) with diverse substitution patterns regarding positions 5, 6, 7 and 8 were synthesised in this thesis. The resulting quinolone amides were evaluated for their antitrypanosomal activity.
Voluminous residues in position C-5 resulted in diminished activities (compounds 13, 16 and 18) and solely small-sized moieties were tolerated. In particular the fluorine atom in position 5 revealed beneficial trypanocidal effects as shown for compounds 6 (IC50 = 0.05 µM), 8 (IC50 = 0.04 µM), and 24 (IC50 = 0.02 µM). Furthermore, having fluorine only in position 5 of the quinolone core could considerably reduce the cytotoxic effects (CC50 >100 µM, SI = >2000 for 6). Hence, the 5-fluoro-substituted quinolone amides were considered superior to GHQ168.
Regarding the C-6 position all other moieties (e.g., H in 9, OCH3 in 10, CF3 in 12) except of a fluorine atom decreased the activity against Trypanosoma brucei brucei. A double fluorination in C-6 and C-8 was not beneficial (IC50 = 0.06 µM for 7) and a single fluorine atom in C-8 even showed a negative effect (IC50 = 0.79 µM for 5).
The logP value is considered a surrogate parameter for lipophlicity and thus, affecting permeability and solubility processes. In particular the fluorine atom influences the lipophilicity due to versatile effects: Lipophilicity is increased by additional fluorine atoms on aromatic rings (7, 23) and reduced by fluorine atoms at an alkyl chain (49), respectively. Additionally, the 5-fluoro-substituted quinolone amides (6, 8, and 24) could prove the contrary effect of decreasing lipophilicity when the aromatic fluorine substituent is in vicinity to a carbonyl group.
For the most promising drug candidates 6, 23, and 24 the respective metabolites and the metabolic turnover were investigated by C. Erk. In comparison to GHQ168 the hydroxylation of the benzylamide was prevented by the para-fluorine atom. Hence, half-life was extended for compound 23 (t1/2 = 6.4 h) and N-desalkylation was the predominant pathway. Moreover, the respective fluorine substitution pattern of the quinolone core affected the metabolism of compound 6. The 5-fluoro-substituted quinolone amide was less prone for biotransformation (t1/2 = 7.2 h) and half-life could even be further prolonged for compound 24 (t1/2 = 7.7 h).
Due to the most appropriate safety profile of compound 6, this particular drug candidate was considered for in vivo study. Its poor solubility made a direct intraperitoneal administration unfeasible. Thus, an amorphous solid dispersion of 6 was generated using the spray-drying method according to the previous protocol. Unfortunately, the required solubility for the predicted in vivo study was not achieved.
Furthermore, the compound class of the quinolone amide was evaluated for its ability for brain penetration. The methanesulfonyl precursor 48 was synthesised and subsequently radiofluorinated in the group of Prof. Dr. Samnick (Department of Nuclear Medicine, University Hospital of Würzburg). The labelled compound [18F]49 was administered to mice, and its distribution throughout the body was analysed using positron emission tomography and autoradiography, respectively. The autoradiography of the murine brains revealed medium to high concentrations of [18F]49. Therefore, the quinolone amides are generally suitable for treating Human African Trypanosomiasis stage II.
A scaffold hopping approach was performed starting from the quinolone amides and concluding with the compound class of pyrazoloquinolin-3-ones. The intramolecular hydrogen bond between the sec. amide and the C-4 carbonyl moiety was replaced by a covalent bond. The two compound classes were comparable regarding the antitrypanosomal activity to some degree (IC50 = 7.9 µM (EK02) vs. 6.37 µM (53a)). However, a final evaluation of 59 was not possible due to poor solubility.
Assay and impurity profiling of the pharmaceuticals are the key routine quality control methods employed worldwide for which High Performance Liquid Chromatography (HPLC) is the most widely used technique. The ability to carry out these routine laboratory procedures in low- and middle- income countries (LMICs) need the methods to be based upon simple instruments manageable with moderate levels of personnel skill and costs involved.
Simple, convenient, and cost effective reverse phase HPLC methods were developed using phosphate buffer and methanol as mobile phase with C18 column as stationary phase for the impurity profiling and assay of beta lactam antibiotics. Isocratic elution and UV detection was employed in these methods. Impurity profiling method was developed for coamoxiclav tablets and ceftriaxone bulk drug. The method for ceftriaxone included a supplementary method to quantify one of its known impurity (Impurity D of ceftriaxone). This method involved use of acetonitrile where as the two main methods were achieved on the targeted method design, described above. With the exception of impurity A of ceftriaxone, the methods developed can successfully quantify impurities to the concentration as low as ≤0.05%, which is in accordance with the current guidelines for the impurity profiling of antibiotics issued by European Medicines Agency.
As ensuring cost reduction was one of the key objectives of carrying out the method development exercise, in situ methods for the preparation of impurities were also identified and some new methods were introduced. The stability of beta lactam antibiotics and the choice of solvent were given due attention during the process of method development revealing information on the presence of new impurities. Deacetyl cefotaxime and 2-mercaptobenzathiazole were identified in this process as new impurities of ceftriaxone currently not listed under known impurities by United States Pharmacopoeia and European Pharmacopoeia. However, deacetyl cefotaxime is a known impurity of cefotaxime whereas the latter molecule is a degradation product of one of the synthesis impurities of ceftriaxone. This substance is reported to be carcinogenic and is resolved using the supplementary method developed for ceftriaxone, hence making its detection and quantification possible. A known inactive impurity of ceftriaxone (Impurity A, E-isomer of ceftriaxone) was` also shown to be produced by exposure to day light, thus warranting the light protection of the ceftriaxone solution, an information that is of critical importance in the clinical settings.
A series of experimentation was carried out on the finished products of beta lactam antibiotics sampled from Pakistan and few other countries, to identify key quality issues in the samples. Though the limited sample size and convenient sampling did not provide results that could yield a decisive figure for the country status for prevalence of substandard and falsified medical products, but the experiments have clearly indicated that the problems in drug quality do exist and beta lactam antibiotics form a class of high-risk medicine with respect to surveillance for poor-quality medicines. Isolation of unknown impurities was also carried out along with the introduction of new and modified methods for preparation of impurities of beta-lactam antibiotics.
In addition, detailed literature survey was carried out for understanding the complex problem of the poor-quality medicine, impact of poor quality antimicrobials on health care system and the magnitude of the problem at the global level. The country status of Pakistan regarding quality of medicines was recorded based upon the available documentary evidence. The current technologies and strategic options available for low- and middle-income countries in aiding fight for combating poor quality medicines was also laid down to design recommendations for Pakistan. A comprehensive review of the information technology tools used for identification and control of substandard and falsified medicines was also conducted.
In dieser Arbeit wurde ein Verfahren zur effizienten Herstellung von (−)-trans-Cannabidiol (CBD, 10), (−)-trans-Δ9-Tetrahydrocannabinol (Dronabinol, 21) und (−)-trans-Cannabidivarin (CBDV, 30) durch kontinuierliche Synthese untersucht und entwickelt.
CBD konnte durch kontinuierliche Synthese in drei Schritten aus Olivetolcarbonsäuremethylester (OM, 6) und Menthadienol G (3) mit einer Ausbeute von 41 % synthetisiert werden. Bei optimierten Bedingungen betrug die Reinheit nach Kristallisation > 99 %. Die Stereochemie konnte durch Röntgenstrukturanalyse eindeutig als 1R,6R bestimmt werden. Vorteilhaft war dabei, dass Toluol anstatt eines chlorierten Lösungsmittels verwendet werden konnte. Weitere Vorteile waren die kurze Reaktionszeit und die Tatsache, dass die Synthese bei Raumtemperatur durchgeführt werden konnte. Es konnten fünf Nebenprodukte detektiert und identifiziert werden, wovon eines Dronabinol war.
Bei optimierten Reaktionsparametern konnte eine Ausbeute an Dronabinol von 64,5 % erreicht werden. Durch Simulated Moving Bed (SMB)-Chromatographie konnte Dronabinol kontinuierlich mit einem Gehalt von > 95 % hergestellt werden. Nach der Synthese waren vier Verunreinigungen detektierbar, und zwar Olivetol (17), CBD, Exo-Tetrahydrocannabinol (Exo-THC, 23) und Δ8-Tetrahydrocannabinol (Δ8-THC, 22). Durch die SMB-Aufreinigung konnten alle Verunreinigungen auf einen monographiekonformen (USP 37) Gehalt abgereichert werden. Nach der finalen destillativen Aufarbeitung trat eine noch nicht identifizierte Verunreinigung in einem Gehalt von ca. 0,4 Flächen-% auf.
CBDV konnte durch kontinuierliche Synthese in drei Schritten aus Divarincarbonsäuremethylester (DM, 25) und Menthadienol G synthetisiert werden. Die Ausbeute betrug ca. 30 %, die Reinheit nach Kristallisation > 99 %. Es konnten fünf Nebenprodukte detektiert werden, die im Rahmen dieser Arbeit nicht weiter charakterisiert wurden.
Der Syntheseweg bietet durch Modifikation der Seitengruppen an Position 6 (R1) und Position 5 (R2) der Alkylbenzol-Gruppe Zugang zu synthetischen Cannabinoiden mit einem CBD- oder CBDV-Grundgerüst. Es wurden neun neue Cannabinoide hergestellt: 2-Hydroxyethylcannabidiolat (2-HEC, 31), 2-Hydroxypentylcannabidiolat (2 HPC, 32), Glycerylcannabidiolat (GCBD, 33), Cyclohexylcannabidiolat (CHC, 34), Hexylcannabidiolat (HC, 35), N-Methylsulfonylcannabidiolat (NMSC, 36), 2 Hydroxyethylcannabidivarinolat (2-HECBDV, 37), Cyclohexylcannabidivarinolat (CHCBDV, 38) und Hexylcannabidivarinolat (HCBDV, 39).
Die Bindungsaffinität wurde in Cannabinoid-Rezeptor-transfizierten HEK293EBNA-Zellen untersucht, die intrinsische Aktivität in CHO-Zellen, die Induktion von NF-κB (nuclear factor kappa B) sowie von NFAT (nuclear factor of activated T cells) in Jurkat-T Zellen, die Induktion proinflammatorischer Zytokine und Chemokine (Interleukin(IL)-6, IL-1β, CC Chemokinligand 2' (CCL2) und Tumornekrosefaktor(TNF)-α) auf mRNA-Ebene in RAW264.7-Makrophagen und die Expression von proinflammatorischen Zytokinen (IL-1β, IL-6, IL-8, TNF-α) und Prostaglandin E2 (PGE2) auf Proteinebene in primären humanen Monozyten.
Die CBD-Derivate zeigten eine höhere Selektivität für CB2-Rezeptoren. Die CBDV-Derivate HCBDV und CHCBDV zeigten eine spezifische Bindung an CB1- und CB2-Rezeptoren im nanomolaren Bereich. 2-HEC, 2-HPC, GCBD und NMSC wirkten als Agonisten an CB2- und als Antagonisten am CB1-Rezeptor. CHC band an CB1 und CB2 im submikromolaren Bereich und schien ein Agonist für beide Rezeptoren zu sein. 2- HECBD wirkte als Agonist auf CB2-Rezeptoren und als Antagonist auf CB1-Rezeptoren. In Jurkat-T Zellen hemmte NMSC dosisabhängig die Aktivität von NF-κB sowie von NFAT. 2-HEC, 2-HPC und GCBD hemmten die Expression von NFAT ebenfalls dosisabhängig. CHC und HC reduzierten dosisabhängig die Expression von IL-1β- und CCL2-mRNA in RAW264.7-Makrophagen. NMSC hemmte in geringeren Dosen IL-1β, CCL2 sowie TNF-α und induzierte in höheren Dosen einen starken Anstieg der IL-6-mRNA. In primären humanen Monozyten hemmten 2 HEC und GCBD konzentrationsabhängig die Synthese von IL-1β, IL-6 und TNF-α. 2-HPC hemmte dosisabhängig die Bildung von TNF-α und IL-6. HC verminderte dosisabhängig die Freisetzung von TNF-α und IL-6. NMSC steigerte die durch LPS erhöhte Freisetzung von IL-1β noch weiter, hemmte aber TNF-α, IL-8 und PGE2.
Die hier untersuchten CBD- und CBDV-Derivate sind geeignet, gezielt an Cannabinoid-Rezeptoren zu wirken. Einige der Derivate könnten als selektive CB2-Agonisten genutzt werden. Die Länge des aliphatischen Rests an R2 von CBD (Pentyl-Cannabinoiden) und CBDV (Propyl-Cannabinoiden) korrelierte nicht mit der Bindungsaffinität. Eine höhere Polarität an R1 (2-HECBDV > NMSC > GCBD > 2-HEC) schien demgegenüber die agonistische Aktivität an CB2 zu begünstigen. Um den Ergebnissen zur Beziehung zwischen Struktur und Wirkung noch mehr Bedeutung zu geben, wären weitere synthetische Derivate und deren Testung notwendig.
Functionalization of cells, extracellular matrix components and proteins for therapeutic application
(2019)
Glycosylation is a biochemical process leading to the formation of glycoconjugates by linking glycans (carbohydrates) to proteins, lipids and various small molecules. The glycans are formed by one or more monosaccharides that are covalently attached, thus offering a broad variety depending on their composition, site of glycan linkage, length and ramification. This special nature provides an exceptional and fine tunable possibility in fields of information transfer, recognition, stability and pharmacokinetic. Due to their intra- and extracellular omnipresence, glycans fulfill an essential role in the regulation of different endogenous processes (e.g. hormone action, immune surveillance, inflammatory response) and act as a key element for maintenance of homeostasis. The strategy of metabolic glycoengineering enables the integration of structural similar but chemically modified monosaccharide building blocks into the natural given glycosylation pathways, thereby anchoring them in the carbohydrate architecture of de novo synthesized glycoconjugates. The available unnatural sugar molecules which are similar to endogenous sugar molecules show minimal perturbation in cell function and - based on their multitude functional groups - offer the potential of side directed coupling with a target substance/structure as well as the development of new biological properties. The chemical-enzymatic strategy of glycoengineering provides a valuable complement to genetic approaches.
This thesis primarily focuses on potential fields of application for glycoengineering and its further use in clinic and research. The last section of this work outlines a genetic approach, using special Escherichia coli systems, to integrate chemically tunable amino acids into the biosynthetic pathway of proteins, enabling specific and site-directed coupling with target substances. With the genetic information of the methanogen archaea, Methanosarcina barkeri, the E. coli. system is able to insert a further amino acid, the pyrrolysine, at the ribosomal site during translation of the protein. The natural stop-codon UAG (amber codon) is used for this newly obtained proteinogenic amino acid.
Chapter I describes two systems for the integration of chemically tunable monosaccharides and presents methods for characterizing these systems. Moreover, it gives a general overview of the structure as well as intended use of glycans and illustrates different glycosylation pathways. Furthermore, the strategy of metabolic glycoengineering is demonstrated. In this context, the structure of basic building blocks and the epimerization of monosaccharides during their metabolic fate are discussed.
Chapter II translates the concept of metabolic glycoengineering to the extracellular network produced by fibroblasts. The incorporation of chemically modified sugar components in the matrix provides an innovative, elegant and biocompatible method for site-directed coupling of target substances. Resident cells, which are involved in the de novo synthesis of matrices, as well as isolated matrices were characterized and compared to unmodified resident cells and matrices. The natural capacity of the matrix can be extended by metabolic glycoengineering and enables the selective immobilization of a variety of therapeutic substances by combining enzymatic and bioorthogonal reaction strategies. This approach expands the natural ability of extracellular matrix (ECM), like the storage of specific growth factors and the recruitment of surface receptors along with synergistic effects of bound substances. By the selection of the cell type, the production of a wide range of different matrices is possible.
Chapter III focuses on the target-oriented modification of cell surface membranes of living fibroblast and human embryonic kidney cells. Chemically modified monosaccharides are inserted by means of metabolic glycoengineering and are then presented on the cell surface. These monosaccharides can later be covalently coupled, by “strain promoted azide-alkyne cycloaddition“ (SPAAC) and/or “copper(I)-catalyzed azide-alkyne cycloaddition“ (CuAAC), to the target substance. Due to the toxicity of the copper catalysator in the CuAAC, cytotoxicity analyses were conducted to determine the in vivo tolerable range for the use of CuAAC on living cell systems. Finally, the efficacy of both bioorthogonal reactions was compared.
Chapter IV outlines two versatile carrier – spacer – payload delivery systems based on an enzymatic cleavable linker, triggered by disease associated protease. In the selection of carrier systems (i) polyethylene glycol (PEG), a well-studied, Food and Drug Administration approved substance and very common tool to increase the pharmacokinetic properties of therapeutic agents, was chosen as a carrier for non-targeting systems and (ii) Revacept, a human glycoprotein VI antibody, was chosen as a carrier for targeting systems. The protease sensitive cleavable linker was genetically inserted into the N-terminal region of fibroblast growth factor 2 (FGF-2) without jeopardizing protein activity. By exchanging the protease sensitive sequence or the therapeutic payload, both systems represent a promising and adaptable approach for establishing therapeutic systems with bioresponsive release, tailored to pre-existing conditions.
In summary, by site-specific functionalization of various delivery platforms, this thesis establishes an essential cornerstone for promising strategies advancing clinical application. The outlined platforms ensure high flexibility due to exchanging single or multiple elements of the system, individually tailoring them to the respective disease or target site.
Sacha inchi oil is a premier raw material with highly nutritional and functional features for the foodstuff, pharmaceutical, beauty, and personal care industries. One of the most important facts about this oil is the huge chemical content of unsaturated and polyunsaturated fatty acids. However, the current available information on the characterization of the triglyceride composition and the advance physicochemical parameters relevant to emulsion development is limited. Therefore, this research focused on providing a detailed description of the lipid composition using high-resolution tandem mass spectrometry and thorough physicochemical characterization to find the value of the required hydrophilic–lipophilic balance (HLB). For this, a study in the interfacial tension was evaluated, followed by the assessment of different parameters such as creaming index, droplet size, viscosity, zeta potential, pH, and electrical conductivity for a series emulsified at thermal stress condition. The results show that fatty acids are arranged into glycerolipids and the required HLB to achieve the maximum physical stability is around 8.
In the „Position Paper of the Division of Clinical Pharmacy of the German Pharmaceutical Society (DPhG)” clinical pharmacy is defined as the science and practice of the rational use of drugs1, which includes the individualization of drug therapy. Clinical pharmacists therefore need a profound knowledge of the pharmacokinetic properties of relevant drugs, and clinical factors that are influencing these properties.
Against the background of individualizing drug therapy, pharmacokinetic and clinical factors are studied in this thesis.
In order to obtain an overview of the existing data on the pharmacokinetics of imipenem / cilastatin and meropenem in critically ill patients, a literature review for each of these carbapenem antibiotics was performed. These reviews included studies in critically ill patients as well as studies in healthy volunteers. While the reported results of studies in healthy volunteers had a small variability, studies in critically ill patients show significant differences in the resulting pharmacokinetics. These differences were not only between, but also within these studies, resulting in a high variability of the pharmacokinetic parameters of the carbapenems in critically ill patients. Furthermore, the results of studies in critically ill patients indicate that clinical factors and in particular renal function have different effects on the pharmacokinetics of imipenem and cilastatin.
A therapeutic drug monitoring (TDM) program for antibiotics was initiated in an intensive care unit. The calculation of the pharmacokinetics of imipenem / cilastatin and meropenem was carried out with a population pharmacokinetic approach (POP-PK) and in addition with a non-compartmental approach (NCA).
The POP-PK analysis showed that the pharmacokinetics of imipenem and cilastatin could be described adequately with a 1-compartment model. The resulting mean total body clearance (CL) of imipenem and cilastatin was 11.6 L/h (4.24 to 27.5) and 6.14 L/h (0.520 to 26.6 L/h). The nonrenal clearance was estimated to be 5.30 L / h (24.9% CV) for imipenem and 0.138 L / h (33.3% CV) for cilastatin.
The results of the NCA were in good agreement with the results of the POP-PK approach, as the NCA resulted in an imipenem clearance of 15.5 ± 7.3 L / hr and cilastatin clearance of 10.1 ± 9.9 L / h. The individual clearances resulting from the different pharmacokinetic approaches were in good correlation showing correlation coefficients (r) of 0.882 (p <0.001) and 0.908 (p <0.001) for imipenem and cilastatin.
In summary, this study identified and quantified significant differences between the individual clearance mechanisms of imipenem and cilastatin. This is particularly true for patients with impaired renal function and sepsis. As imipenem / cilastatin is only available in a fixed dose combination, those patients might be treated inadequately with this combination. The great variability in the pharmacokinetics of imipenem and cilastatin in septic patients underscores the importance of a TDM program of both substances.
For meropenem, a PK/PD model was developed that predicts the concentration gradients of meropenem, serum creatinine, C-reactive protein and procalcitonin simultaneously. A non-linear relationship between the clearance of creatinine and meropenem was identified and the resulting equation for the calculation of the total body clearance of meropenem (for a 70 kg patient) was: 0.480 L/h + 9.86 L/h. (CLCR/6L/h)0.593, with 0.480 L/h representing the nonrenal clearance of meropenem.
The resulting mean meropenem clearance of the NCA was 11.9 ± 8.7 L/h. The individual clearances resulting from the different pharmacokinetic approaches were poorly correlated showing a correlation coefficient (r) of 0.502 (p <0.001).
In summary, this study showed a non-linear relationship of meropenem clearance and creatinine clearance. The model shows that the renal function may change rapidly and to a significant extent in patients with sepsis and septic shock, which in turn, underscores that creatinine concentrations are not in steady state in these patients. Conversely, dose adjustment based on creatinine values might lead to inappropriate therapy. This underlines the importance of a TDM program for meropenem in critically ill patients.
The two most important considerations when choosing an antibiotic for the prophylaxis of postoperative bone infections are its activity against the whole spectrum of bacteria, which might be involved in bone infections, and its ability to penetrate bone tissue and thus to achieve concentrations above the minimum inhibitory concentration (MIC) of the corresponding pathogens.
In order to gain information on this data, a study was conducted which investigated the pharmacokinetics of ampicillin / sulbactam in plasma, cortical and cancellous bone. Pharmacokinetic parameters in plasma were determined using NCA. The bone penetration represents the ratio of the concentration in the bone tissue to plasma concentration at the time of bone removal. The resulting half-life of ampicillin and sulbactam in plasma was 1.60 0.37 h and 1.70 0.42 h. The elimination of both substances was in a good correlation with creatinine clearance and resulted in correlation coefficients (r) of 0.729 (p = 0.003) for ampicillin and 0.699 (p = 0.005) for sulbactam. The mean clearance and the mean volume of distribution of ampicillin and sulbactam were 10.7 3.9 and 10.3 3.3 L/h, and 23.9 7.9 and 24.3 6.8 L. The mean concentrations of ampicillin in the cortical and cancellous bone were 6.60 4.22 and 10.15 7.40 µg/g, resulting in bone penetration ratios of 9.1 5.7 and 16.2 16.9 %. For sulbactam the corresponding concentrations were 3.91 2.52 and 5.73 4.20 µg/g, resulting in bone penetration ratios of 10.6 6.3 and 17.5 16.1 %.
In summary, this study shows that the bone penetration of both substances is on average rather unsatisfactory and has a high variability, which can lead to inadequate bone concentrations for the prophylaxis of bone infections. One factor that could be identified for the penetration of both substances into cancellous bone was the period between the application of the drug and the removal of the bone. Therefore, a time interval between the administration of the antibiotic and the incision should be considered.
Immunosuppression is a risk factor for the development of various malignancies, including hematologic diseases. While the relationship between the use of immunosuppressive therapy with methotrexate and the development of an Epstein-Barr virus (EBV) associated lymphoproliferative disease (LPD) has been well established, this connection is less evident for immunosuppressive therapy with azathioprine.
The patient presented by us was immunosuppressed with azathioprine for autoimmune hepatitis. The development of an EBV-associated Hodgkin-like lymphoma under this immunosuppressive therapy and especially the regression of the lymphoma after cessation of azathioprine confirms the relationship between this immunosuppressant, EBV-infection and the development of Hodgkin-like lymphoma. Therefore, albeit in rare cases, azathioprine-related lymphomas may respond to mere cessation of immunosuppressive therapy without need for chemotherapy.
Apart from viral infections, drugs are a major cause of acute liver failure. Due to the lack of specific symptoms or tests, it is difficult to diagnose a drug-induced liver injury. We report a case of a young patient in whom different antibiotics, the analgesic and antipyretic acetaminophen or a combination of these drugs may have led to DILI resulting in life-threatening ALF. Based on this case report, we describe a procedure to exclude non-drug related causes and discuss the hepatotoxic potential of the involved drugs in this case.
Detailed insight into the internal structure of drug‐loaded polymeric micelles is scarce, but important for developing optimized delivery systems. We observed that an increase in the curcumin loading of triblock copolymers based on poly(2‐oxazolines) and poly(2‐oxazines) results in poorer dissolution properties. Using solid‐state NMR spectroscopy and complementary tools we propose a loading‐dependent structural model on the molecular level that provides an explanation for these pronounced differences. Changes in the chemical shifts and cross‐peaks in 2D NMR experiments give evidence for the involvement of the hydrophobic polymer block in the curcumin coordination at low loadings, while at higher loadings an increase in the interaction with the hydrophilic polymer blocks is observed. The involvement of the hydrophilic compartment may be critical for ultrahigh‐loaded polymer micelles and can help to rationalize specific polymer modifications to improve the performance of similar drug delivery systems.