Institut für Pharmazie und Lebensmittelchemie
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During the last decades the number of biologics increased dramatically and several biopharmaceutical drugs such as peptides, therapeutic proteins, hormones, enzymes, vaccines, monoclonal antibodies and antibody-drug conjugates conquered the market. Moreover, administration and local delivery of growth factors has gained substantial importance in the field of tissue engineering. Despite progress that has been made over the last decades formulation and delivery of therapeutic proteins is still a challenge. Thus, we worked on formulation and delivery strategies of therapeutic proteins to improve their biological performance.
Phase I of this work deals with protein stability with the main focus on a liquid protein formulation of the dimeric fusion protein PR-15, a lesion specific platelet adhesion inhibitor. In order to develop an adequate formulation ensuring the stability and bioactivity of PR-15 during storage at 4 °C, a pH screening, a forced degradation and a Design of Experiments (DoE) was performed. First the stability and bioactivity of PR-15 in 50 mM histidine buffer in relation to pH was evaluated in a short-term storage stability study at 25 °C and 40 °C for 4 and 8 weeks using different analytical methods. Additionally, potential degradation pathways of PR-15 were investigated under stressed conditions such as heat treatment, acidic or basic pH, freeze-thaw cycles, light exposure, induced oxidation and induced deamidation during the forced degradation study. Moreover, we were able to identify the main degradation product of PR-15 by performing LC/ESI-MS analysis. Further optimization of the injectable PR 15 formulation concerning pH, the choice of buffer and the addition of excipients was studied in the following DoE and finally an optimal PR-15 formulation was found.
The growth factors BMP-2, IGF-I and TGF-β3 were selected for the differentiation of stem cells for tissue engineering of cartilage and bone in order to prepare multifunctionalized osteochondral implants for the regeneration of cartilage defects.
Silk fibroin (SF) was chosen as biomaterial because of its biocompatibility, mechanical properties and its opportunity for biofunctionalization. Ideal geometry of SF scaffolds with optimal porosity was found in order to generate both tissues on one scaffold.
The growth factors BMP-2 and IGF-I were modified to allow spatially restricted covalent immobilization on the generated porous SF scaffolds. In order to perform site-directed covalent coupling by the usage of click chemistry on two opposite sides of the scaffold, we genetically engineered BMP-2 (not shown in this work; performed by Barbara Tabisz) and IGF-I for the introduction of alkyne or azide bearing artificial amino acids. TGF β3 was immobilized to beads through common EDC/NHS chemistry requiring no modification and distributed in the pores of the entire scaffold.
For this reason protein modification, protein engineering, protein immobilization and bioconjugation are investigated in phase II. Beside the synthesis the focus was on the characterization of such modified proteins and its conjugates. The field of protein engineering offers a wide range of possibilities to modify existing proteins or to design new proteins with prolonged serum half-life, increased conformational stability or improved release rates according to their clinical use.
Site-directed click chemistry and non-site-directed EDC/NHS chemistry were used for bioconjugation and protein immobilization with the aim to underline the preferences of site-directed coupling.
We chose three strategies for the incorporation of alkyne or azide functionality for the performance of click reaction into the protein of interest: diazonium coupling reaction, PEGylation and genetic engineering. Azido groups were successfully introduced into SF by implementation of diazonium coupling and alkyne, amino or acid functionality was incorporated into FGF-2 as model protein by means of thiol PEGylation. The proper folding of FGF-2 after PEGylation was assessed by fluorescence spectroscopy, WST-1 proliferation assay ensured moderate bioactivity and the purity of PEGylated FGF-2 samples was monitored with RP-HPLC. Moreover, the modification of native FGF-2 with 10 kDa PEG chains resulted in enhanced thermal stability.
Additionally, we genetically engineered one IGF-I mutant by incorporating the unnatural amino acid propargyl-L-lysine (plk) at position 65 into the IGF-I amino acid sequence and were able to express hardly verifiable amounts of plk-IGF-I. Consequently, plk-IGF-I expression has to be further optimized in future studies in order to generate plk-IGF-I with higher yields.
Bioconjugation of PEGylated FGF-2 with functionalized silk was performed in solution and was successful for click as well as EDC/NHS chemistry. However, substantial amounts of unreacted PEG-FGF-2 were adsorbed to SF and could not be removed from the reaction mixture making it impossible to expose the advantages of click chemistry in relation to EDC/NHS chemistry. The immobilization of PEG-FGF-2 to microspheres was a trial to increase product yield and to remove unreacted PEG-FGF-2 from reaction mixture. Bound PEG-FGF-2 was visualized by fluorescence imaging or flow cytometry and bioactivity was assessed by analysis of the proliferation of NIH 3T3 cells. However, immobilization on beads raised the same issue as in solution: adsorption caused by electrostatic interactions of positively charged FGF-2 and negatively charged SF or beads. Finally, we were not able to prove superiority of site-directed click chemistry over non-site-directed EDC/NHS.
The skills and knowledge in protein immobilization as well as protein characterization acquired during phase II helped us in phase III to engineer cartilage tissue in biofunctionalized SF scaffolds.
The approach of covalent immobilization of the required growth factors is relevant because of their short in vivo half-lives and aimed at controlling their bioavailability. So TGF-β3 was covalently coupled by means of EDC/NHS chemistry to biocompatible and biostable PMMA beads. Herein, we directly compared bioactivity of covalently coupled and adsorbed TGF-β3. During the so-called luciferase assay bioactivity of covalent coupled as well as adsorbed TGF-β3 on PMMA beads was ensured. In order to investigate the real influence of EDC/NHS chemistry on TGF-β3’s bioactivity, the amount of immobilized TGF-β3 on PMMA beads was determined. Therefore, an ELISA method was established. The assessment of total amount of TGF-β3 immobilized on the PMMA beads allowed as to calculate coupling efficiency. A significantly higher coupling efficiency was determined for the coupling of TGF-β3 via EDC/NHS chemistry compared to the reaction without coupling reagents indicating a small amount of adsorbed TGF-β3. These results provide opportunity to determine the consequence of coupling by means of EDC/NHS chemistry for TGF β3 bioactivity. At first sight, no statistically significant difference between covalent immobilized and adsorbed TGF-β3 was observed regarding relative luciferase activities. But during comparison of total and active amount of TGF-β3 on PMMA beads detected by ELISA or luciferase assay, respectively, a decrease of TGF-β3’s bioactivity became apparent. Nevertheless, immobilized TGF β3 was further investigated in combination with SF scaffolds in order to drive BMSCs to the chondrogenic lineage. According to the results obtained through histological and immunohistochemical studies, biochemical assays as well as qRT-PCR of gene expression from BMSCs after 21 days in culture immobilized TGF-β3 was able to engineer cartilage tissue. These findings support the thesis that local presentation of TGF β3 is superior towards exogenous TGF β3 for the development of hyaline cartilage. Furthermore, we conclude that covalent immobilized TGF β3 is not only superior towards exogenously supplemented TGF-β3 but also superior towards adsorbed TGF-β3 for articular hyaline cartilage tissue engineering. Diffusion processes were inhibited through covalent immobilization of TGF-β3 to PMMA beads and thereby a stable and consistent TGF-β3 concentration was maintained in the target area.
With the knowledge acquired during phase II and III as well as during the studies of Barbara Tabisz concerning the expression and purification of plk-BMP-2 we made considerable progress towards the formation of multifunctionalized osteochondral implants for the regeneration of cartilage defects. However, further studies are required for the translation of these insights into the development of multifunctionalized osteochondral SF scaffolds.
Alzheimer’s disease is a complex network of several pathological hallmarks. These characteristics always occur concomitantly and cannot be taken as distinct features of the disease. While there are hypotheses trying to explain the origin and progression of the illness, none of them is able to pinpoint a definitive cause. This fact challenges researchers not to focus on one individual hallmark but, bearing in mind the big picture, target two or more indications at once. This work, therefore, addresses two of the major characteristics of AD: the cholinergic hypothesis and neurotoxic oxidative stress. The former was achieved by targeting the postsynaptic muscarinic M1 acetylcholine receptor to further investigate its pharmacology, and the latter with the synthesis of neuroprotective natural antioxidant hybrids.
The first aim was the design and synthesis of dualsteric agonists of the muscarinic M1 acetylcholine receptor. Activation of this receptor was previously shown to improve AD pathologies like the formation of Aβ and NFTs and protect against oxidative stress and caspase activation. Selectively targeting the M1 receptor is difficult as subtypes M1 – M5 of the muscarinic AChRs largely share the same orthosteric binding pocket. Orthosteric ligands are thus unsuitable for selective activation of one specific subtype. Secondary, allosteric binding sites are more diverse between subtypes. Allosteric ligands are, however, in most cases dependent on an orthosteric ligand to cause downstream signals. Dualsteric ligands thus utilize the characteristics of both orthosteric and allosteric ligands in form of a message-address concept. Bridged by an alkylene-linker, the allosteric part ensures selectivity, whereas the orthosteric moiety initiates receptor activation. Two sets of compounds were synthesised in this sense. In both cases, the orthosteric ligand carbachol is connected to an allosteric ligand via linkers of different chain length. The first set utilizes the selective allosteric M1 agonist TBPB, the second set employs the selective M1 positive allosteric modulator BQCA. Six compounds were obtained in twelve-step syntheses each. For each one, a reference compound lacking the carbachol moiety was synthesised. The dualsteric ligands 1a-c and 2a c were tested in the IP1 assay. The assay revealed that the TBPB-dualsterics 1 are not able to activate the receptor, whereas the respective TBPB-alkyl reference compounds 27 gave signals depending on the length of the alkylene-linker, suggesting allosteric partial agonism of alkyl compounds 27 and no dualsteric binding of the putatively dualsteric compounds 1. The dualsteric BQCA molecules 2, however, activated the receptor as expected. Efficacy of the C5 linked compound 2b was the highest, yet C3 and C8 compounds (2a and 2c) also showed partial agonism. In this case, the reference compounds 31 showed no receptor activation, implying the intended dualsteric binding mode of the BQCA-carbachol compounds 2. Further investigations will be conducted by the working group of Dr. Christian Tränkle at the Department of Pharmacology at the University of Bonn to confirm binding modes and determine affinities as well as selectivity of the synthesised dualsteric compounds.
The second project dealt with the design, synthesis and biological evaluation of neuroprotective esters of the flavonolignan silibinin. While silibinin is already a potent antioxidant, it has been observed that the 7-OH group has a pro-oxidative character, making this position attractive for functionalisation. In order to obtain more potent antioxidants, the pro-oxidative position was esterified with other antioxidant moieties like ferulic acid 35 and derivatives thereof. Seventeen esters of silibinin 32, including pure diastereomers of 7 O feruloylsilibinin (43a and 43b) and a cinnamic acid ester of 2,3-dehydrosilibinin 46, were synthesised by regioselective esterification using acyl chlorides under basic conditions. The physicochemical antioxidant properties were assessed in the FRAP assay. This assay revealed no improvement of the antioxidant properties except for 7-O-dihydrosinapinoylsilibinin 39b. These results, however, do not correlate with the neuroprotective properties determined in the HT-22 hippocampal neuronal cell model. The assay showed overadditive neuroprotective effects of the esters exceeding those of its components and equimolar mixtures with the most potent compounds being 7-O-cinnamoylsilibinin 37a, 7-O-feruloylsilibinin 38a and the acetonide-protected caffeic acid ester 40a. These potent Michael system bearing compounds may be considered as “PAINS”, but the assays used to assess antioxidant and neuroprotective activities were carefully chosen to avoid false positive readouts. The most potent compounds 37a and 38a, as well as the diastereomers 43a and 43b, were further studied in assays related to AD. In vitro ischemia, inhibition of microglial activation, PC12 cell differentiation and inhibition of Aβ42 and τ protein aggregation assays showed similar results in terms of overadditive effects of the synthesised esters. Moreover, the diastereomers 43a and 43b showed differences in their activities against oxytosis (glutamate-induced apoptosis), inhibition of Aβ42 and τ protein aggregation, and PC12 cell differentiation. The stereospecific effect or mode of action against Aβ42 and τ protein aggregation is more pronounced than that of silybin A (32a) and silybin B (32b) reported in literature and needs to be elucidated in future work. Stability measurements in cell culture medium revealed that the esters do not only get hydrolysed but are partially oxidised to their respective 2,3-dehydrosilibinin esters. Because dehydrosilibinin 45 itself is described as a more potent antioxidant than silibinin 32, 7 O cinnamoyl-2,3-dehydrosilibinin 46 was expected to be even more potent than its un-oxidised counterpart 37a in terms of neuroprotection. The oxytosis assay, however, showed that the neurotoxicity of 46 is much more pronounced, especially at higher concentrations, reducing its neuroprotective potential. Dehydrosilibinin esters are therefore inferior to the silibinin esters for application as neuroprotectants, because of the difficulty of their synthesis and their increased neurotoxicity. A synergistic effect of both species (silibinin and the oxidised form) might, however, be possible or even necessary for the pronounced neuroprotective effects of silibinin esters. As the dehydro-species show distinct neuroprotective properties at low concentrations, their continuous formation over time might make an essential contribution to the overall neuroprotection of the synthesised esters. Due to solubility issues for some of the ester compounds, 7-O-cinnamoylsilibinin 37a was converted into a highly soluble hemisuccinate. The vastly improved solubility of 7 O cinnamoyl-23-O-succinylsilibinin 48 was confirmed in shake-flask experiments. Contrary to expectation, stability examinations showed that the succinyl compound 48 is not cleaved to form 7-O-cinnamoylsilibinin 37a. Neuroprotection assays confirmed that 48 is not a prodrug of the corresponding ester. It was determined that the main site of hydrolysis is the 7-position, cleaving 37 to silibinin 32 and cinnamic acid thus reducing the compound’s neuroprotective effects. Nevertheless, the compound still showed neuroprotection at a concentration of 25 µM. The improved solubility might be more beneficial than the higher neuroprotection of the poorly soluble parent compound 37a in vivo. 7 O Cinnamoylsilibinin 37a was further investigated to reduce Aβ25 35 induced learning impairment in mice. While tendencies of improved short-term and long-term memory in the animals were observed, the effects are not yet statistically significant in both Y-maze and passive avoidance tests. A greater number of test subjects is necessary to ensure correctness of the preliminary results presented in this work. However, an effect of ester 37a is observable in vivo, showing blood-brain barrier penetration. The esters synthesised are a novel approach for the treatment of AD as they show strong neuroprotective effects and their hydrolysis products or metabolites are only non-toxic natural products.