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Progressive loss of skeletal muscle mass, strength and function poses a major threat to independence and quality of life, particularly in the elderly. To date, sarcopenia therapy consists of resistance exercise training in combination with protein supplementation due to the limited efficacy of available pharmacological options in counteracting the effects of muscle wasting. Therapeutic intervention with growth factors including insulin-like growth factor I (IGF-I) or inhibitors of myostatin a potent suppressor of myogenesis hold potential to rebalance the altered activity of anabolic and catabolic cytokines. However, dosing limitations due to acute side effects and disruptions of the homeostasis have so far precluded clinical application.
Intending to provide a therapy with a superior safety and efficacy profile by directing drug release to inflamed tissue and minimizing off-target activity, we designed bioresponsive delivery systems for an anti-catabolic peptide and anabolic IGF-I responding to local flares of muscle wasting.
In Chapter I, current concepts for bioorthogonal conjugation methods are discussed and evaluated based on various drug delivery applications. With a focus on protein delivery, challenges and potential pitfalls of each chemical and enzymatic conjugation strategy are analyzed and opportunities regarding their use for coupling of biomolecules are given. Based on various studies conjugating proteins to polymers, particles and biomaterials using different site-directed approaches, the chapter summarizes available strategies and highlights certain aspects requiring particular consideration when applied to biomolecules. Finally, a decision process for selection of an optimum conjugation strategy is exemplarily presented.
Three of these bioorthogonal coupling reactions are applied in Chapter II detailing the potential of site-directed conjugation in the development of novel, homogenous drug delivery systems. The chapter describes the design of a delivery system of a myostatin inhibitor (MI) for controlled and local release counteracting myositis flares. MI release from the carrier is driven by increased matrix metalloproteinase (MMP) levels in compromised muscle tissues cleaving the interposed linker, thereby releasing the peptide inhibitor from the particulate carrier. Release experiments were performed to assess the response towards various MMP isoforms (MMP-1, -8, -9 and -13) – as upregulated during skeletal muscle myopathies – and the release pattern of the MI in case of disease progression was analyzed. By selection of the protease-sensitive linker (PSL) showing variable susceptibilities to proteases, release rates of the MI can be controlled and adapted. Immobilized MI as well as released MI as response to MMP upregulation was able to antagonize the effects of myostatin on cell signalling and myoblast differentiation.
The approach of designing bioresponsive protein delivery systems was also applied to the anabolic growth factor IGF-I, as described in Chapter III. Numerous studies of PEGylated proteins or peptides reveal, that successful therapy is challenged by safety and efficacy issues, as polymer attachment considerably alters the properties of the biologic, thereby jeopardizing clinical efficacy. To this end, a novel promising approach is presented, intending to exploit beneficial effects of PEGylation on pharmacokinetics, but addressing the pharmacodynamic challenges by releasing the protein upon entering the target tissue. This was realized by integration of a PSL between the PEG moiety and the protein. The soluble polymer conjugate was produced by site-directed, enzymatic conjugation of IGF-I to the PSL, followed by attachment of a 30 kDa-PEG using Strain-promoted azide-alkyne cycloaddition (SPAAC). This strategy illustrates the potential of bioorthogonal conjugation (as described in Chapter I) for generation of homogenous protein-polymer conjugates with reproducible outcome, but also emphasizes the altered protein properties resulting from permanent polymer conjugation. As compared to wild type IGF-I, the PEGylated protein showed considerable changes in pharmacologic effects – such as impaired insulin-like growth factor binding protein (IGFBPs) interactions, submaximal proliferative activity and altered endocytosis patterns. In contrast, IGF-I characteristics were fully restored upon local disintegration of the conjugate triggered by MMP upregulation and release of the natural growth factor.
For successful formulation development for the proteins and conjugates, the careful selection of suitable excipients is crucial for a safe and reliable therapy. Chapter IV addresses one aspect by highlighting the chemical heterogeneity of excipients and associated differences in performance. Polysorbate 80 (PS80) is a surfactant frequently used in protein formulations to prevent aggregation and surface adsorption. Despite being widely deployed as a standard excipient, heterogeneous composition and performance entails the risk of eliciting degradation and adverse effects on protein stability. Based on a comprehensive study using different batches of various suppliers, the PS80 products were characterized regarding chemical composition and physicochemical properties, facilitating the assessment of excipient performance in a formulation. Noticeable deviations were recorded between different suppliers as well as between batches of the same suppliers. Correlation of all parameters revealed, that functionality related characteristics (FRCs) could be reliably predicted based on chemical composition alone or by a combination of chemical and physicochemical properties, respectively.
In summary, this thesis describes and evaluates novel strategies for the targeted delivery and controlled release of biologics intended to counteract the imbalance of anabolic and catabolic proteins observed during aging and musculoskeletal diseases. Two delivery platforms were developed and characterized in vitro – (i) using anti-catabolic peptides immobilized on a carrier for local delivery and (ii) using soluble IGF-I polymer conjugates for systemic application. Both approaches were implemented by bioorthogonal coupling strategies, which were carefully selected in consideration of limitations, side reactions and efficiency aspects. Bioresponsive release of the active biomolecules following increased protease activity could be successfully realized. The therapeutic potential of these approaches was demonstrated using various cell-based potency assays. The systems allow targeted and controlled release of the growth factor IGF-I and anti-catabolic peptides thereby overcoming safety concerns of current growth factor therapy and thus positively impacting the benefit-risk profile of potent therapeutics. Taking potential heterogeneity and by-product concerns into account, comprehensive excipient characterization was performed and a predictive algorithm for FRCs developed, in order to facilitate formulation design and guarantee a safe and efficient therapy from start to finish.
Renin–angiotensin system (RAS) plays an important role in the regulation of blood pressure and hormonal balance. Using positron emission tomography (PET) technology, it is possible to monitor the physiological and pathological distribution of angiotensin II type 1 receptors (AT\(_1\)), which reflects the functionality of RAS. A new \(^{18}\)F-labeled PET tracer derived from the clinically used AT\(_1\) antagonist valsartan showing the least possible chemical alteration from the valsartan structure has been designed and synthesized with several strategies, which can be applied for the syntheses of further derivatives. Radioligand binding study showed that the cold reference FV45 (K\(_i\) 14.6 nM) has almost equivalent binding affinity as its lead valsartan (K\(_i\) 11.8 nM) and angiotensin II (K\(_i\) 1.7 nM). Successful radiolabeling of FV45 in a one-pot radiofluorination followed by the deprotection procedure with 21.8 ± 8.5% radiochemical yield and >99% radiochemical purity (n = 5) enabled a distribution study in rats and opened a path to straightforward large-scale production. A fast and clear kidney uptake could be observed, and this renal uptake could be selectively blocked by pretreatment with AT\(_1\)-selective antagonist valsartan. Overall, as the first \(^{18}\)F-labeled PET tracer based on a derivation from clinically used drug valsartan with almost identical chemical structure, [\(^{18}\)F]FV45 will be a new tool for assessing the RAS function by visualizing AT\(_i\) receptor distributions and providing further information regarding cardiovascular system malfunction as well as possible applications in inflammation research and cancer diagnosis.
While life expectancy increases worldwide, treatment of neurodegenerative diseases such as AD becomes a major task for industrial and academic research. Currently, a treatment of AD is only symptomatical and limited to an early stage of the disease by inhibiting AChE. A cure for AD might even seem far away. A rethinking of other possible targets is therefore necessary. Addressing targets that can influence AD even at later stages might be the key. Even if it is not possible to find a cure for AD, it is of great value for AD patients by providing an effective medication. The suffering of patients and their families might be relieved and remaining years may be spent with less symptoms and restrictions.
It was shown that a combination of hCB2R agonist and BChE inhibitor might exactly be a promising approach to combat AD. In the previous chapters, a first investigation of dual-acting compounds that address both hCB2R and BChE was illustrated (figure 6.1).
A set of over 30 compounds was obtained by applying SARs from BChE inhibitors to a hCB2R
selective agonist developed by AstraZeneca. In a first in vitro evaluation compounds showed
selectivity over hCB1R and AChE. Further investigations could also prove agonism and showed
that unwanted off-target affinity to hMOP receptor could be designed out. The development of
a homology model for hCB2R (based on a novel hCB1R crystal) could further elucidate the
mode of action of the ligand binding. Lastly, first in vivo studies showed a beneficial effect of
selected dual-acting compounds regarding memory and cognition.
Since these first in vivo studies mainly aim for an inhibition of the BChE, it should be the aim
of upcoming projects to proof the relevance of hCB2R agonism in vivo as well. In addition,
pharmacokinetic as well as solubility studies may help to complete the overall picture.
Currently, hybrid-based dual-acting hCB2R agonists and selective BChE inhibitors are under
investigation in our lab. First in vitro evaluations showed improved BChE inhibition and
selectivity over AChE compared to tacrine.78 Future in vitro and in vivo studies will clarify their
usage as drug molecules with regard to hepatotoxicity and blood-brain barrier penetration.
Since the role of hCB2R is not yet completely elucidated, the use of photochromic toolcompounds
becomes an area of interest. These tool-compounds (and their biological effect) can
be triggered upon irradiation with light and thus help to investigate time scales and ligand
binding.
A set of 5-azobenzene benzimidazoles was developed and synthesized. In radioligand binding
studies, affinity towards hCB2R could be increased upon irradiation with UV-light (figure 6.2).
This makes the investigated compounds the first GPCR ligands that can be activated upon
irradiation (not vice versa).
The aim of upcoming research will be the triggering of a certain intrinsic activity by an
“efficacy-switch”. For this purpose, several attempts are currently under investigation: an
introduction of an azobenzene moiety at the 2-position of the benzimidazole core already led to
a slight difference in efficacy upon irradiation with UV light. Another approach going on in our
lab is the development of hCB1R switches based on the selective hCB1R inverse agonist
rimonabant. First in vitro results are not yet available (figure 6.3).
Diese Arbeit befasst sich mit der Untersuchung des Metabolismus sowie der Reaktivität verschiedener Wirk- und Arzneistoffe mittels flüssigchromatographischer und massen-spektrometrischer Methoden, sie gliedert sich dabei in vier Projekte. Zur Bestimmung des Metabolitenprofils wurde ein passendes In-vitro-Inkubationssystem mit Cytochrom-P-450-Systemen entwickelt. So wurden der Metabolismus und die Pharmakokinetik der Mip-Inhibitoren SF110, SF235 und SF354 gegen Legionellen, sowie neuer antitrypanosomaler Verbindungen MB209, MB343 und MB444 und von Daptomycin bestimmt. Darüber hinaus wurde die antibakterielle Aktivität des Daptomycins gegenüber einem unbekannten Staphylokokkus-Stammes S. sciuri ermittelt. Außerdem wurden Reaktivitätsuntersuchungen neu synthetisierter Inhibitoren gegen Tuberkulose und S. aureus durchgeführt.
Die untersuchten Mip-Inhibitoren lieferten ein Metabolitenprofil, welches durch Ester- und Amidhydrolysen sowie Hydroxylierungen geprägt wurde. Die Verbindung SF110 schien dabei bereits eine gewisse Instabilität der Esterbindung aufzuweisen, da auch im Blindwert entsprechende Spaltprodukte identifiziert werden konnten. Die Hauptmetabolite von SF235 und SF354 bildeten sich durch unterschiedliche Hydrolysen, da die Spaltung des Moleküls von den jeweiligen Substituenten abhängig ist. Innerhalb dieser Substanzklasse dominiert die mikrosomale Enzymkatalyse, da der größte metabolische Umsatz sowie die meisten Metabolite mittels mikrosomaler Fraktion des Menschen bzw. der Maus gefunden wurden. Die Klasse der Mip-Inhibitoren wird somit vor allem durch Cytochrom-P-450-Enzyme umgesetzt, wobei die Hydrophilie durch Einführung polarer OH-Gruppen der Moleküle erhöht wird. Die Hydroxylierung scheint dabei positionsspezifisch, bedingt durch sterische Hinderungen oder dirigierende Einflüsse, abzulaufen. Stabilitätsvergleiche zwischen SF110, SF235 und SF354 zeigten, dass die Einführung einer Amidbindung anstelle der korrespondierenden Esterbindung die Substanzklasse maßgeblich metabolisch stabilisiert. Im Rahmen des murinen In-vivo-Metabolismus wurde beobachtet, dass SF235 einem deutlich stärkeren Metabolismus unterlag als SF354 und sich der Metabolismus vor allem innerhalb der ersten 30 min vollzog. Demgegenüber zeigten die In-vitro-Ergebnisse gegenteilige Ergebnisse, bei denen SF354 die am stärksten metabolisierte Substanz war. Diese widersprüchlichen Ergebnisse deuten darauf hin, dass In-vitro-Modelle nur als Anhaltspunkt verwendet werden sollten, um mögliche Trends abzuleiten.
Metabolismusstudien der Chinolonamide, die gegen die afrikanische Schlafkrankheit wirken sollen, veranschaulichten, dass die größte enzymatische Umsetzung aller drei getesteten Verbindungen mittels cytosolischer Fraktion erfolgte. Die Enzymreaktionen werden vermutlich durch ALDH bzw. MAO dominiert und nicht durch CYP bzw. FMO. Die gebildeten Metabolite in den verschiedenen Fraktionen unterlagen (ω-1)-Oxidationen, N-Desalkylierungen, Amidhydrolysen und aromatischen Hydroxylierungen. Auffallend war, dass eine Hydroxylierung am aromatischen Benzylring nur erfolgen konnte, sofern der Benzylaromat keinen Fluorsubstitutenten trug, da dieser desaktivierend wirkte. Die aromatische Hydroxylierung am Chinolonamid erfolgte dagegen bei allen drei Substanzen. Es wurde somit lediglich eine Hydroxylierung am Benzylring von MB343 festgestellt. Die enzymatische Aktivität aller Substanzen folgte einer Reaktionskinetik 1. Ordnung. Die unterschiedlichen Stabilitäten der Substanzen zeigten einen deutlichen Trend: MB209 wurde, da es die instabilste Verbindung darstellt, im größten Maße umgesetzt, gefolgt von den stabileren Derivaten MB343 und MB444. Die Untersuchung der enzymatischen Aktivitäten zeigte, dass die drei Substanzen, verglichen mit der Leitstruktur GHQ168, eine um den Faktor zehn geringere Aktivität aufwiesen [19]. Aufgrund der eingeführten Fluoratome weisen die Substanzen somit eine wesentlich höhere Stabilität auf. Diese Ergebnisse wurden durch die Untersuchung der Halbwertszeit bestätigt, bei der MB444 den höchsten Wert besaß. Weiterhin ist die Position des Fluorsubstituenten am Chinolongerüst ausschlaggebend für die metabolische Stabilität, wobei MB444 aufgrund des para-Fluorsubstituenten am Chinolonamid die stabilste Verbindung darstellt.
Durch Inkubation von Daptomycin mit unterschiedlichen S. sciuri-Isolaten wurde ein möglicher Inaktivierungsmechanismus beobachtet, bei dem das Antibiotikum durch Spaltung des cyclischen Aminosäureringes, durch Deacylierung des Fettsäureschwanzes, einer Kombination beider Mechanismen oder durch eine Spaltung des heteroaromatischen Ringsystems von Tryptophan inaktiviert wurde. Die Proteasen des Daptomycin-resistenten S. sciuri-Isolats TS92 führten zu einem Daptomycinabbau von 35 %, unabhängig von der eingesetzten Menge des Arzneistoffes. Das Ausmaß des Abbaus scheint darüber hinaus vom eingesetzten Inkubationsmedium abhängig zu sein, da die Proteasen voraussichtlich auf ein bestimmtes Nährmedium angewiesen sind. Der sensitive S. sciuri-Stamm TS93 lieferte die höchste Abbaurate an Daptomycin mit 55 % und widerlegt damit die Vermutung, dass Daptomycin die geringste antibakterielle Aktivität gegenüber diesem S. sciuri-Stamm aufweist. Im In-vitro-Metabolismus zeigte Daptomycin insgesamt eine sehr geringe Umsetzungsmenge mit maximal 5 % nach 4 h und einer geringen Metabolitenbildung. Hier wurde nur ein Metabolit gefunden, welcher auch mittels S. sciuri-Inkubation identifiziert wurde. Dieser Mechanismus könnte somit auf anderem Wege verlaufen.
Die Reaktivitätsstudien der kovalenten Inhibitoren der FadA5-Thiolase gegen Tuberkulose zeigten, dass nur die Verbindungen C1 und C4 eine Reaktivität gegenüber der Aminosäure Cystein93 im aktiven Zentrum besaßen, die somit für den gewünschten Einsatzzweck geeignet sein könnten. Weiterhin wurde bei den kovalenten Inhibitoren der Enoyl-ACP-Reduktase mit dem Enzym FabI, welches im aktiven Zentrum ein Tyrosin besitzt, keine Reaktion festgestellt, da keine Addukte identifiziert wurden. Dies ist vermutlich auf die Unlöslichkeit im verwendeten TRIS-Puffer zurückzuführen.
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.
Although the prevalence of substandard and counterfeit pharmaceutical products is a global problem, it is more critical in resource-constrained countries. The national medicines regulatory authorities (MNRA) in these countries have limited resources to cater for regular quality surveillance programmes aimed at ensuring that medicines in circulation are of acceptable quality. Among the reasons explained to hinder the implementation of these strategies is that compendial monographs are too complicated and require expensive infrastructures in terms of environment, equipment and consumables. In this study it was therefore aimed at developing simple, precise, and robust HPLC and HPTLC methods utilizing inexpensive, readily available chemicals (methanol and simple buffers) that can determine the APIs, other API than declared one, and which are capable of impurity profiling. As an outcome of this study, three isocratic and robust HPLC and two HPTLC methods for sulfadoxine, sulfalene, pyrimethamine, primaquine, artesunate, as well as amodiaquine have been developed and validated. All HPLC methods are operated using an isocratic elution mode which means they can be implemented even with a single pump HPLC system and standard C18 columns. The densitometric sulfadoxine/sulfalene and pyrimethamine method utilizes standard TLC plates as well as inexpensive, readily available and safe chemicals (toluene, methanol, and ethyl acetate), while that for artesunate and amodiaquine requires HPTLC plates as well as triethylamine and acetonitrile due to challenges associated with the analysis of amodiaquine and poorly the detectable artesunate. These HPTLC methods can be implemented as alternative to those requiring HPLC equipment e.g. in countries that already have acquired densitometer equipment. It is understood that HPTLC methods are less sensitive, precise and accurate when compared to HPLC methods, but this hindrance can easily be addressed by sending representative samples to third party quality control laboratories where the analytical results are verified using compendial HPLC methods on a regular basis.
It is therefore anticipated that the implementation of these methods will not only address the problem of limited resources required for medicines quality control but also increase the number of monitored targeted antimalarial products as well as the number of resource- constrained countries participating in quality monitoring campaigns. Moreover, the experiences and skills acquired within this work will be applied to other API groups, e. g. antibiotics, afterwards.
1 Verlängerung der kardialen Repolarisationsdauer unter psychiatrischer Medikation bei gleichzeitigem genetischen Basisrisiko
Vielen Psychopharmaka wird eine repolarisationsverlängernde Wirkung zugeschrieben. Diese unerwünschte Arzneimittelwirkung, erkennbar an einer Verlängerung des QT-Intervalls im Elektrokardiogramm, ist in den vergangenen Jahren, aufgrund des Zusammenhanges mit lebensbedrohlichen Torsades-de-Pointes-Tachyarrhythmien, in den Fokus der klinischen Forschung gerückt. Aufgrund dieser Nebenwirkung werden viele gut wirksame Arzneimittel einer erneuten eingehenden Nutzen-Risiko-Analyse unterzogen und in manchen Fällen führte dies zu einer Limitierung der pharmakologischen Möglichkeiten.
Als Hauptmechanismus für eine Psychopharmaka-induzierte QT-Zeit-Verlängerung gilt die Blockade von kardialen Kaliumkanälen. Aber auch genetische Veränderungen unterschiedlicher kardialer Ionenkanäle gelten als Risikofaktoren, ebenso wie Effekte anderer ionenabhängiger Signalwege. Da Patienten mit genetischer Prädisposition ein defacto erhöhtes Risiko für eine pharmakologisch induzierte QT-Zeit-Verlängerung aufweisen, spricht man von reduzierter Repolarisationsreserve, mit erhöhtem Basislinienrisiko für kardiale Nebenwirkungen.
Ziel war es, über einen additiven genetischen Risikoscore eine Quantifizierung individueller Vulnerabilität zu erreichen und zu zeigen, dass dieses Risiko durch die Kontrolle von Medikamenten-Serumspiegeln modulierbar sein kann.
Aus einer prospektiven Studie, mit 2062 an endogener Psychose leidenden Patienten des Zentrums für Psychische Gesundheit des Universitätsklinikums Würzburg, wurden 392 Patienten (mittleres Alter bei Studieneinschluss 41,0 ± 15,0 Jahre, 36,2 % Frauen) rekrutiert. Primäres Einschlusskriterium für die angeknüpfte, retrospektive Studie war das Vorliegen einer Serumspiegelbestimmung der psychiatrischen Medikation binnen drei Tagen vor oder nach einer elektrokardiographischen Untersuchung (N = 392). Die den Einschlusskriterien entsprechenden 392 Patienten wurden daraufhin auf 62 Einzelpolymorphismen, die in Verbindung mit einer verlängerten QT-Zeit stehen, getestet und die Ergebnisse mit den patientenspezifischen Daten aus den elektrokardiographischen Untersuchungen korreliert.
Des Weiteren wurden, basierend auf vier großen Publikationen des internationalen „Cardiac Safety Consortium“ (77-79, 148), bekannte polygene Risikoscores, die diese Risikopolymorphismen enthalten, anhand des eigenen Patientenkollektivs berechnet und durch Korrelation mit der QT-Zeit überprüft. Diese Scores funktionieren jeweils nach einem Additionsmodell, bei dem nach unterschiedlicher Gewichtung das individuelle Risiko, das durch das Vorhandensein eines bekannten Risikopolymorphismus quantifizierbar wird, zu einem Gesamtrisiko aufsummiert wird.
Darüber hinaus ist das Patientenkollektiv auf einen Zusammenhang zwischen dem Serumspiegel der psychiatrischen Medikation und der QT-Zeit geprüft worden. Dazu wurde das Gesamtkollektiv in medikamentenspezifische Subgruppen unterteilt (Amitriptylin (N = 106), Clomipramin (N = 48), Doxepin (N = 53), Mirtazapin (N = 45), Venlafaxin (N = 50), Aripiprazol (N = 56), Clozapin (N = 127), Haloperidol (N = 41), Olanzapin (N = 37), Perazin (N = 47), Quetiapin (N = 119) und Risperidon (N = 106)).
Abschließend wurden die Subkollektive in einem kombinierten Rechenmodell daraufhin geprüft, ob Zusammenhänge zwischen den genetischen Risikoscores nach Strauss et al. (148) mit dem jeweiligen Medikamenten-Serumspiegel auf die QT-Zeit bestehen.
13 der 62 untersuchten Einzelpolymorphismen zeigten einen signifikanten Zusammenhang mit einer verlängerten Repolarisationsdauer. Ebenfalls korrelieren polygene Risikoscores einer verlängerten kardialen Repolarisation und erklären einen dabei signifikanten Anteil der Varianz. Die Ergebnisse der Literatur, bezüglich der Scores nach Pfeufer et al. (77) (R = 0,124, p = 0,014; N = 392), nach Noseworthy et al. (79) (R = 0,169; p = 0,001; N = 392), sowie nach Strauss et al. (148) (R = 0,199; p = 0,000; N = 392) konnten anhand des eigenen Kollektives reproduziert werden, wohingegen der Score von Newton-Cheh et al. (78) keinen signifikanten Zusammenhang mit der QT-Zeit zeigte (R = 0,029; p = 0,568; N = 392).
In der Subgruppenanalyse konnte ein stark vom Serumspiegel abhängiger, verlängernder Effekt auf die QT-Zeit für die Arzneistoffe Amitriptylin, Nortriptylin, Clomipramin, und Haloperidol nachgewiesen werden. Die Analyse der mit Amitriptylin behandelten Patienten (N = 106) ergab für Nortriptylin (F (1,104) = 5.986; p = .016, R = .233), als auch für den Summenspiegel aus Amitriptylin und Nortriptylin (F (1,104) = 4.408, p = .038, R = .202) einen signifikanten, nach Cohen einen mittelstarken Zusammenhang mit der QT-Zeit. Starke Effekte auf die QT-Zeit wurden im Zusammenhang mit den Serumspiegeln der Medikamente Clomipramin (F (1,46) = 39.589, p < .001, R = .680, N = 48) und Haloperidol (F (1,39) = 12.672, p = .001, korrigiertes R2= .245, N = 41) errechnet.
Ein kombiniertes Rechenmodell, das sowohl den Einfluss des jeweiligen Serumspiegels, als auch des genetischen Risikoscores nach Strauss et al. (148) berücksichtigte, erlaubte bei diesen Arzneistoffen eine signifikant höhere Varianzaufklärung der QT-Zeit, als die jeweiligen Effekte für sich genommen.
Die QT-Zeit gilt als erwiesenermaßen genauso abhängig von der individuellen genetischen Ausstattung, wie auch von Serumspiegeln potentiell als QT-verlängernd eingestufter Medikamente. Diese Effekte scheinen additiv verknüpfbar, so dass das von Roden et al. entwickelte Konzept der reduzierten Repolarisationsreserve (54) als bestätigt gelten darf. Die jeweiligen Einzeleffekte vom genetischen Risiko, sowie der Medikation haben zusammen einen größeren Einfluss auf die gemessenen QT-Zeit als für sich alleine genommen. Durch die Genetik lässt sich somit tatsächlich eine grobe vorab-Risikoabschätzung treffen. Dies könnte nach sorgfältiger Nutzen-Risiko-Analyse durch Kontrollen des EKGs und des Serumspiegels moduliert werden und somit vielfältigere therapeutische Möglichkeiten erhalten.
2 Entwicklung und Validierung einer Dried-Blood-Spot-Methode zum therapeutischen Drug Monitoring von Clozapin und Quetiapin
Die Technik der Extraktion und Analyse von Stoffen aus getrocknetem Blut ist bereits seit den 1960er Jahren bekannt, wurde bis zur jüngeren Vergangenheit aber eher zu diagnostischen Zwecken angewendet. Durch Fortschritte in der Analytik im Sinne ausgefeilterer Chromatographie und sensitiverer Detektion wurde das Verfahren der Dried-Blood-Spot-Analytik auch für die Spiegelbestimmung von Arzneistoffen interessant. So wurden auch im Bereich des Therapeutischen Drug Monitorings bereits Methoden, beispielsweise für Antibiotika, Antiepileptika, Virostatika und in jüngerer Zeit auch Antidiabetika publiziert. Die Vorteile in der Probenhandhabung und durch geringeren Aufwand bei der Blutentnahme sowie geringeres Probenentnahmevolumen werden durch weitere Fortschritte im Bereich der Analytik vordergründiger.
Ziel war es, ein Extraktionsverfahren zu entwickeln und zu validieren, dass die gemeinsame Quantifizierung der häufig verabreichten Antipsychotika Clozapin und Quetiapin aus einem einzelnen getrockneten Blutstropfen ermöglicht.
Die Extraktion mit einer Mischung aus 99 % Acetonitril und 1 % 1 M Salzsäure und anschließender HPLC-Analyse mit Säulenschaltung und photometrischer Detektion wurde nach den Richtlinien der Gesellschaft für toxikologische und forensische Chemie (GTFCh) (146) validiert. Sie entsprach sämtlichen Anforderungen bezüglich Linearität, Bestimmungsgrenze, Stabilität, Genauigkeit, Extraktionsausbeute und Robustheit.
Somit gilt diese Methode in der Praxis als anwendbar und dürfte, nach Überprüfung der therapeutischen Bereiche für kapillares Vollblut im Vergleich zu den bereits definierten Bereichen für venöse entnommene Serumproben, Eingang in die klinische Praxis finden.
In resent years the rate of biologics (proteins, cytokines and growth-factors) as newly registered drugs has steadily risen. The greatest challenge for pharmaceutical biologics poses its arrival at the desired target location due to e.g. proteolytic and pH dependent degradation, plasma protein binding, insolubility etc. Therefore, advanced drug delivery systems, where biologics are site directed immobilized to carriers mimicking endogenous storage sites such as the extra cellular matrix can enormously assist the application and consequently the release of exogenous administered pharmaceutical biologics. We have resorted to the fibroblast growth factor 2/ heparansulfate/ fibroblast growth factor bindingprotein 1 system as a model.
Phase I deals with the selection and subcloning of a wild type murine FGF-2 construct into the bacterial pHis-Trx vector system for high yields of expression and fast, feasible purification measurements. This first step enables the provision of mFGF-2, which plays a pivotal part as a growth factor in the wound healing process as well as the vascularization of tumors, for future investigations. Therefore, the correct expression of mFGF-2 was monitored via MALDI-MS and SDS-PAGE, whereas the proper folding of the tertiary beta-trefoil structure was assessed by fluorescence spectroscopy. The MTT assay allowed us to ensure that the bioactivity was comparable to sourced FGF-2. In the last step, the purity; a requirement for future binding- and protein-protein interaction assays was monitored chromatographically (RP-HPLC). In addition, a formulation for freeze-drying was developed to ensure protein stability and integrity over a period of 60 days. Altogether, the bacterial expression and purification proved to be suitable, leading to bioactive and stable production of mFGF-2.
In Phase II the expression, purification and characterization of FGFBP1, as the other key partner in the FGF-2/ HS/ FGFBP1 system is detailed. As FGFBP1 exhibits a complex tertiary structure, comprised of five highly conserved disulfide bonds and presumably multiple glycosylation sites, a eukaryotic expression was used. Human embryonic kidney cells (HEK 293F) as suspension cells were transiently transfected with DNA-PEI complexes, leading to expression of Fc-tagged murine FGFBP1. Different PEI to DNA ratios and expression durations were investigated for optimal expression yields, which were confirmed by western blot analysis and SDS-PAGE. LC-MS/MS analysis of trypsin and elastase digested FGFBP1 gave first insights of the three O-glycosylation sites. Furthermore, the binding protein was modified by inserting a His6-tag between the Fc-tag (for purification) and the binding protein itself to enable later complexation with radioactive 99mTc as radio ligand to track bio distribution of administered FGFBP1 in mice. Overall, expression, purification and characterization of mFGFBP1 variants were successful with a minor draw back of instability of the tag free binding protein.
Combining the insights and results of expressed FGF-2 as well as FGFBP1 directed us to the investigation of the interaction of each partner in the FGF-2/ HS/ FGFBP1 system as Phase III. Thermodynamic behavior of FGF-2 and low molecular weight heparin (enoxaparin), as a surrogate for HS, under physiological conditions (pH 7.4) and pathophysiological conditions, similar to hypoxic, tumorous conditions (acidic pH) were monitored by means of isothermal titration calorimetry. Buffer types, as well as the pH influences binding parameters such as stoichiometry (n), enthalpy (ΔH) and to some extent the dissociation constant (KD). These findings paved the way for kinetic binding investigations, which were performed by surface plasmon resonance assays. For the first time the KD of full length FGFBP1 and FGF-2 was measured. Furthermore the binding behavior of FGF-2 to FGFBP1 in the presence of various heparin concentrations suggest a kinetic driven release of bound FGF-2 by its chaperone FGFBP1.
Having gathered multiple data on the FGF-2 /HS /FGFBP1 system mainly in solution, our next step in Phase IV was the development of a test system for immobilized proteins. With the necessity to better understand and monitor the cellular effects of immobilized growth factors, we decorated glass slides in a site-specific manner with an RGD-peptide for adhesion of cells and via the copper(I)-catalyzed-azide-alkyne cycloaddition (CuAAC) a fluorescent dye (a precursor for modified proteins for click chemistry). Human osteosarcoma cells were able to grow an the slides and the fluorescence dye was immobilized in a biocompatible way allowing future thorough bioactivity assay such as MTT-assays and phospho-ERK-assays of immobilized growth factors.
A major problem regarding public health is the emergence of antibiotic resistant bacterial strains, especially methicillin resistant Staphylococcus aureus (MRSA). This is mainly attributed to the unnecessary overuse of antimicrobial drugs by patients; however, one aspect that is often neglected is their untargeted mechanism of action, affecting not only the infection itself but also commensal bacteria which are often opportunistic pathogens causing many diseases as well. Therefore, our goal was to develop a bioresponsive antibiotic delivery system triggered by virulence factors. The designed system is comprised of a polymer to enhance its pharmacokinetic profile, a peptide cleavable linker, and the antibiotic agent itself. The bacterial protease aureolysin which is expressed by S. aureus during infections would cleave the linker and partially release the antibiotic which would be still attached to a remaining tetrapeptide. These would be cleaved by a group of proteases naturally present in plasma called aminopeptidases, finally releasing the compound.
In the first part of this project, we searched for a suitable sequence to serve as a cleavable linker. It should be sensitive towards the target bacterial protease but not be cleaved by any human enzymes to guarantee the specificity of the system. Therefore, we synthesized three peptide sequences via Solid Phase Peptide Synthesis and incubated them with aureolysin as well as with many human matrix Metalloproteases. The analysis and quantification of enzymatic activity was monitored chromatographically (RP-HPLC). The plasminogen originated sequence was chosen since it was not sensitive towards MMPs, but cleaved by aureolysin.
In the second part, we tried to incorporate the chosen peptide sequences as crosslinkers in hydrogel formulations. The purpose was to physically incorporate the antibiotic within the hydrogel, which would be released by the cleavage of those sequences and the consequent loosening the hydrogel net. For that purpose we used a commercially available hydrogel kit with a PVA matrix modified with maleimide, which allows a conjugation reaction with thiol functionalized crosslinkers. Three fluorophores were chosen to serve as antibiotic models and a diffusion assay was performed. Only the glomerular structured Green Fluorescent Protein (GFP) presented a low diffusion rate, thus the aureolysin release assays were performed only using this prototype. Assays showed that with a low hydrogel polymer concentration, the fluorophore either quickly diffused into the medium or was not released at all. The physical incorporation of the antibiotic within the hydrogel pores was therefore abolished as a suitable release approach. For a second attempt, we covalently bound a fluorophore to the linker, which was conjugated to the hydrogel matrix. The incubation with aureolysin and subsequent RP-HPLC analysis showed a peak with the same retention time correspondent to the fragment product after cleavage of the free linker. This is a proof that the concept of linking the peptide sequence to the antibiotic is a promising strategy for its bioresponsive release.
Within the third part of this study, we analyzed the degradation of the resulted fragment after aureolysin activity and subsequent full release of the antibiotic by human aminopeptidases. We determined the concentration of those enzymes in human plasma and synthesized the fragment by conjugating the tetrapeptide sequence to aminofluorescein via EDC/NHS reaction. By incubating the construct with the lowest aminopeptidase concentration measured in plasma, the fluorophore was completely released within two hours, showing the efficacy of these enzymes as bioresponsive agents.
The last part was the construction of the PEGylated linker-antibiotic. For this purpose we chose the tetracycline like antibiotic chelocardin (CHD) as our prototype. The conjugation of the linker- CHD to the polymer was performed by copper free click chemistry. The cleavage rate of the linker by aureolysin was very similar to the one obtained for the free peptide, indicating that the PEGylation does not interfere on the enzymatic activity. However, by trying to increase the loading ratio of chelocardin onto the polymer, we observed a very low cleavage rate for the system, indicating the formation of aggregates by those constructs.
The designed system has proved to be a smart strategy for the delivery on demand of antibiotics in which the drug is only released by the presence of S. aureus during their virulent state.
Substandard and falsified (SF) medicines have emerged as a global public health issue within the last two decades especially in low- and middle-income countries (LMICs). Serious consequences of this problem include a loss of trust and increased financial costs due to less disease control and more frequent complications during therapy. Of note, antimicrobial resistance is an additional long-term implication of poor-quality antimicrobials. This review covers information technology tools including medicines authentication tools (MAT) as mobile apps and messaging service, 2D barcoding approaches with drug safety alert systems, web based drug safety alerts, radiofrequency identification tags, databases to support visual inspection, digital aids to enhance the performance of quality evaluation kits, reference libraries for identification of falsified and substandard medicines, and quality evaluation kits based on machine learning for field testing. While being easy to access and simple to use, these initiatives are gaining acceptance in LMICs. Implementing 2D barcoding based on end-to-end verification and "Track and Trace" systems has emerged as a step toward global security in the supply chain. A breakthrough in web-based drug safety alert systems and data bases was the establishment of the Global Surveillance and Monitoring System by the World Health Organization in 2013. Future applications include concepts including "lab on a chip" and "paper analytical devices" and are claimed to be convenient and simple to use as well as affordable. The principles discussed herein are making profound impact in the fight against substandard and falsified medicines, offering cheap and accessible solutions.