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The chloroform extract of Valeriana wallichii (V. wallichii) rhizomes was investigated to elucidate the structures responsible for reported antileishmanial activity. Besides bornyl caffeate (1, already been reported by us previously), bioassay-guided fractionation resulted in two additional cinnamic acid derivatives 2–3 with moderate leishmanicidal activity. The structure of a novel nepetolactone derivative 4 having a cinnamic acid moiety was elucidated by means of spectral analysis. To the best of our knowledge villoside aglycone (5) was isolated from this plant for the first time. The bioassay-guided fractionation yielded two new (compounds 6–7) and two known valtrates (compounds 8–9) with leishmanicidal potential against Leishmania major (L. major) promastigotes. In addition, β-bisabolol (10), α-kessyl alcohol (11), valeranone (12), bornyl isovalerate (13) and linarin-2-O-methylbutyrate (14) were identified. This is the first report on the isolation of 4'-demethylpodophyllotoxin (15), podophyllotoxin (16) and pinoresinol (17) in V. wallichii. In total thirteen known and four new compounds were identified from the extract and their cytotoxic and antileishmanial properties were evaluated.
Intraperitoneal adhesions are fibrous bands that connect tissues in the peritoneal cavity that are usually separated. These adhesions form as a consequence of trauma, inflammation or surgical interventions and often result in severe consequences such as chronic pain, small bowel obstructions or female infertility.
The aim of this thesis was to develop a synthetic barrier device for adhesion prevention made of modified poly(lactide) [PLA]. Solid PLA films (SurgiWrap®) are already successfully in clinical use due to the good biocompatibility and the biodegradability of the material resulting in non-toxic degradation products since lactic acid is naturally part of the metabolic circles of the human body. Considering the brittleness and stiffness of the films, the long degradation time of several months as well as the need for suturing, there is potential for optimization. Through a copolymerization with the hydrophilic poly(ethylene glycol) [PEG], a reduction of the degradation time was intendend. Moreover, the copolymerization should also lead to an improvement of the mechanical properties of the films since PEG acts as plasticizer for PLA. Linear PLA-PEG-PLA triblock copolymers as well as star-shaped PEG-PLA copolymers were synthesized via standard ring opening polymerization to tailor the barrier properties. Besides solid films, solution electrospun meshes from PLA and the synthesized PEG-PLA copolymers were investigated for a potential application as well. Since suturing of a barrier additionally induces adhesion formation, alginate coated membranes were prepared in order to achieve self-adhesiveness. With the intention to reduce infections and consequently inflammation, electrospun meshes and solvent cast films were loaded with the antibacterial drug triclosan and drug release as well as antibacterial efficacy was investigated.
Mechanical tests confirmed that through the variation of the PEG content and branching the mechanical properties can be tailored and are in good accordance with the glass transition temperatures [Tg] of the polymers. Consequently, potentially adequate mechanical properties for surgical handling as well as for the performance within the patient’s body were successfully achieved. Degradation studies revealed that the degradation time was significantly shorter for PEG-PLA membranes than for PLA films and with an appropriate PEG content could be adjusted to the intended time frame. Cell adhesion and viability tests confirmed the non-toxicity of the clinically used PLA films as well as of PEG-PLA films and meshes. With a bioadhesion test the benefit of an alginate coated side towards the pure PLA film concerning self-adhesiveness was successfully demonstrated. Moreover, optical evaluations and a T-peel test of different alginate coated PLA films showed that the cohesion between the chemically different layers was distinctly enhanced by the use of an appropriate PEG-PLA mesh as intermediate cohesion promoting layer. In in vitro release studies with triclosan loaded films a higher release was determined for PEG-PLA than for PLA films. In agar diffusion tests a higher and longer inhibition of staphylococcus aureus growth was observed confirming the release results. Moreover, drug loaded meshes (especially drug loaded after electrospinning) showed enhanced and elongated bacterial inhibition in comparison to films.
Charakterisierung der pulmonalen Pharmakokinetik von Salmeterol und Insulin-like Growth Factor-1
(2015)
Für inhalativ applizierte Arzneimittel spielt das Ausmaß der pulmonalen Absorption eine entscheidende Rolle. Für Substanzen, die lokal in der Lunge wirken sollen, sind für eine gute Wirksamkeit hohe lokale Wirkstoffkonzentrationen, und für eine geringe Nebenwirkungsrate niedrige systemische Plasmaspiegel wichtig. Sollen allerdings Substanzen das Lungenepithel überwinden und im systemischen Kreislauf wirken, ist eine hohe systemische Verfügbarkeit für eine gute Wirkung gewünscht. Das Ziel dieser Studie war es mit in vitro und ex vivo Methoden das Absorptions- und Permeationsverhalten von pulmonal applizierten Substanzen zu studieren.
Der Transportmechanismus über das Lungenepithel des langwirksamen ß2-Agonisten Salmeterol wurde mithilfe des humanen ex vivo Lungenperfusionsmodells untersucht. Die Anwendung von L-Carnitin als Hemmstoff von organischen Kationen/Carnitin Transportern (OCT/N) bewirkte eine Verringerung der pulmonalen Absorption von Salmeterol von ca. 90 %, was auf eine Beteiligung von Transportern, möglicherweise des OCTN2 oder OTCN1, für den Transport von Salmeterol über das Lungenepithel hindeutete. Es wurde somit zum ersten Mal erfolgreich gezeigt, dass Salmeterol wahrscheinlich als Substrat der Transportproteine fungiert und der Übertritt über das Lungenepithel von organischen Kationen/Carnitin Transportern abhängig ist. Bisher wurde eine Interaktion von Salmeterol mit den OCT/N nur in in vitro Versuchen studiert und Salmeterol wurde nur als Hemmstoff und nicht als Substrat untersucht. Die Beteiligung eines Transporters für die pulmonale Absorption von Salmeterol steht außerdem im Einklang mit Untersuchungen über weitere ß2-Agonisten wie das kurzwirksame Salbutamol und das langwirksame GW597901. Somit scheinen sowohl lipophile als auch hydrophile ß2-Agonisten Substrate für die OCT/N zu sein.
Die Fähigkeit von IGF-1, nach pulmonaler Applikation in den systemischen Kreislauf zu gelangen, wurde in der vorliegenden Studie mit Hilfe des Lungenperfusionsmodells untersucht. Das IGF-1 wurde gebunden an Trehalose oder an Fibroin als Pulver verabreicht. Die Trehalose sollte eine schnelle Abgabe des IGF 1 bewirken, und das Fibroin sollte zum einen ein Trägermaterial mit schützenden Eigenschaften für das IGF 1 darstellen, und zum anderen sollte eine mögliche verzögerte Freisetzung von IGF-1 aus Fibroin in einem ex vivo Modell untersucht werden, die in vorausgegangenen in vitro Versuchen über 3 h lang vorhanden war. Das Peptid wurde nach der Applikation sowohl der Trehalosepartikel als auch der Fibroinpartikel pulmonal absorbiert und folgte einer linearen Verteilungskinetik. Dieses lineare Absorptionsverhalten des IGF-1 war vergleichbar mit der Kinetik von inhalativem Insulin, die in in vivo Studien beobachtet wurde. Somit konnte gezeigt werden, dass das IGF-1 nach pulmonaler Applikation systemisch verfügbar sein könnte und eine vergleichbare pulmonale Pharmakokinetik wie das strukturell ähnliche Insulin besitzt. Außerdem unterschied sich das Absorptionsverhalten von IGF-1, gebunden an Trehalose, nicht signifikant von dem von IGF-1/Fibroin, was im Gegensatz zu in vitro Untersuchungen stand, in denen das IGF-1 verzögert aus Fibroin freigesetzt wurde. Somit wirkte sich die kontrollierte Abgabe in vitro nicht auf die Verteilungskinetik ex vivo aus. Daraus ergibt sich, dass sowohl Trehalose als auch Fibroin als Trägermaterial für IGF-1 zur pulmonalen Applikation geeignet wären, und dass IGF-1, gebunden an Fibroin eine Formulierung wäre, die zum einen das IGF 1 schützen kann und die zum anderen eine gleiche pulmonale Kinetik wie IGF 1, gebunden an schnell auflösende Trägersubstanzen, besitzt. Außerdem wurde dadurch die Wichtigkeit betont, die Pharmakokinetik von pulmonal verabreichten Substanzen am intakten Organ mit erhaltener Komplexität und Funktionalität zu untersuchen, und dass das Lungenperfusionsmodell hierfür eine geeignete Methode darstellt. Darüber hinaus wurde belegt, dass mithilfe des Lungenperfusionsmodells erfolgreich pharmakokinetische Daten für nieder- und höhermolekulare Substanzen gesammelt werden können, die als Aerosol oder als Pulver appliziert werden.
Auch in den in der vorliegenden Arbeit durchgeführten in vitro Permeationsversuchen, die mit der Bronchialepithelzelllinie Calu-3 durchgeführt wurden, zeigte IGF-1 vergleichbare lineare Permeationseigenschaften wie das Insulin, mit einem apparenten Permeationskoeffizienten von 1,49 * 10-8 cm/sec für IGF-1 und 2,11 * 10-8 cm/sec für Insulin. Das IGF 1 schien durch die Calu-3 Zellen sowohl parazellulär als auch transzytotisch zu permeieren, wie es für Makromoleküle generell vermutet wird. Durch die Verwendung von Hemmstoffen der Transzytose bzw. bestimmter endozytotischer Mechanismen in den Permeationsstudien konnte gezeigt werden, dass, wie bereits genannt, der Transport durch die Zellen eine wichtige Rolle für den Übertritt von IGF-1 über Calu-3 Zellmonolayer spielte. Die Studien ergaben außerdem, dass die zelluläre Aufnahme des IGF-1 unabhängig von Clathrin und abhängig von Dynamin war.
Der Einsatz einer humanen bronchioalveolären Lavage in den Permeationsversuchen bewirkte zum einen eine Erhöhung des Transportes von IGF 1 durch die Calu-3 Zellen, und zum anderen war die zelluläre Aufnahme in diesem Fall unabhängig von Dynamin und unterschied sich somit von den vorherigen Untersuchungen, in denen keine Lavage eingesetzt wurde. Das bedeutet, dass Faktoren in einer bronchioalveolaren Lavage enthalten waren, die sowohl das Ausmaß der Permeation als auch den Mechanismus der zellulären Aufnahme von IGF-1 in Calu-3 Zellen beeinflussten.
Zusammenfassend konnten in der vorliegenden Arbeit erfolgreich weitere Hinweise für die Beteiligung von Transportern an der pulmonalen Absorption von ß2-Agonisten mithilfe des ex vivo Lungenperfusionsmodells gefunden werden, was somit eine wertvolle Ergänzung zu bisher vorhanden in vitro Studien darstellt. Daneben wurde zum ersten Mal gezeigt, dass das IGF-1 nach Applikation in die Lunge pulmonal absorbiert werden könnte. Das belegt den Nutzen der Lunge als Eintrittsort in den systemischen Kreislauf, was vor allem für peptidische Arzneistoffe von Bedeutung ist.
\textbf{Molecular Determinants of Drug-Target Residence Times of Bacterial Enoyl-ACP Reductases.} Whereas optimization processes of early drug discovery campaigns are often affinity-driven, the drug-target residence time $t_R$ should also be considered due to an often strong correlation with \textit{in vivo} efficacy of compounds. However, rational optimization of $t_R$ is not straightforward and generally hampered by the lack of structural information about the transition states of ligand association and dissociation. The enoyl-ACP reductase FabI of the fatty acid synthesis (FAS) type II is an important drug-target in antibiotic research. InhA is the FabI enzyme of \textit{Mycobacterium tuberculosis}, which is known to be inhibited by various compound classes. Slow-onset inhibition of InhA is assumed to be associated with the ordering of the most flexible protein region, the substrate binding loop (SBL). Diphenylethers are one class of InhA inhibitors that can promote such SBL ordering, resulting in long drug-target residence times. Although these inhibitors are energetically and kinetically well characterized, it is still unclear how the structural features of a ligand affect $t_R$.
Using classical molecular dynamics (MD) simulations, recurring conformational families of InhA protein-ligand complexes were detected and structural determinants of drug-target residence time of diphenyl\-ethers with different kinetic profiles were described. This information was used to deduce guidelines for efficacy improvement of InhA inhibitors, including 5'-substitution on the diphenylether B-ring. The validity of this suggestion was then analyzed by means of MD simulations.
Moreover, Steered MD (SMD) simulations were employed to analyze ligand dissociation of diphenylethers from the FabI enzyme of \textit{Staphylococcus aureus}. This approach resulted in a very accurate and quantitative linear regression model of the experimental $ln(t_R)$ of these inhibitors as a function of the calculated maximum free energy change of induced ligand extraction. This model can be used to predict the residence times of new potential inhibitors from crystal structures or valid docking poses.
Since correct structural characterization of the intermediate enzyme-inhibitor state (EI) and the final state (EI*) of two-step slow-onset inhibition is crucial for rational residence time optimization, the current view of the EI and EI* states of InhA was revisited by means of crystal structure analysis, MD and SMD simulations. Overall, the analyses affirmed that the EI* state is a conformation resembling the 2X23 crystal structure (with slow-onset inhibitor \textbf{PT70}), whereas a twist of residues Ile202 and Val203 with a further opened helix $\alpha 6$ corresponds to the EI state. Furthermore, MD simulations emphasized the influence of close contacts to symmetry mates in the SBL region on SBL stability, underlined by the observation that an MD simulation of \textbf{PT155} chain A with chain B' of a symmetry mate in close proximity of the SBL region showed significantly more stable loops, than a simulation of the tetrameric assembly. Closing Part I, SMD simulations were employed which allow the delimitation of slow-onset InhA inhibitors from rapid reversible ligands.
\textbf{Prediction of \textit{Mycobacterium tuberculosis} Cell Wall Permeability.} The cell wall of \textit{M. tuberculosis} hampers antimycobacterial drug design due to its unique composition, providing intrinsic antibiotic resistance against lipophilic and hydrophilic compounds. To assess the druggability space of this pathogen, a large-scale data mining endeavor was conducted, based on multivariate statistical analysis of differences in the physico-chemical composition of a normally distributed drug-like chemical space and a database of antimycobacterial--and thus very likely permeable--compounds. The approach resulted in the logistic regression model MycPermCheck, which is able to predict the permeability probability of small organic molecules based on their physico-chemical properties. Evaluation of MycPermCheck suggests a high predictive power. The model was implemented as a freely accessible online service and as a local stand-alone command-line version.
Methodologies and findings from both parts of this thesis were combined to conduct a virtual screening for antimycobacterial substances. MycPermCheck was employed to screen the chemical permeability space of \textit{M. tuberculosis} from the entire ZINC12 drug-like database. After subsequent filtering steps regarding ADMET properties, InhA was chosen as an exemplary target. Docking to InhA led to a principal hit compound, which was further optimized. The quality of the interaction of selected derivatives with InhA was subsequently evaluated using MD and SMD simulations in terms of protein and ligand stability, as well as maximum free energy change of induced ligand egress. The results of the presented computational experiments suggest that compounds with an indole-3-acethydrazide scaffold might constitute a novel class of InhA inhibitors, worthwhile of further investigation.
Dietary polyphenols have been related to beneficial effects on humans’ health. Pycnogenol®, a dietary polyphenol-rich food supplement complies with the monograph “Maritime pine extract” in the United States Pharmacopeia (USP) and has demonstrated effects in different diseases. Several human trials concerning knee osteoarthritis have shown significant improvement of the symptoms like reducing the pain and the stiffness of the joint(s) upon intake of Pycnogenol®. After oral intake of multiple doses of Pycnogenol® previously low concentrations in the nanomolar range of monomeric extract constituents have been found in human plasma as well as a bioactive metabolite, δ-(3,4-dihydroxy-phenyl)-γ-valerolactone (M1), which is formed by the human intestinal flora from the procyanidins’ catechin units. It is not clear yet which compound(s) of the complex extract is (are) mainly responsible for the described clinical effects of Pycnogenol®. To gain deeper insights into the in vivo fate of the pine bark extract the distribution of its constitutents and metabolites was closer investigated in the present thesis.
Initial in vitro experiments suggested a facilitated cellular uptake of M1 into human erythrocytes, possibly via GLUT-1 transporter. For elucidating further the in vitro and in vivo metabolism of M1 in human blood cells, a metabolomic approach was performed using UPLC-ESI-qTOF-MSE analysis, which revealed a comprehensive and rapid metabolism of M1 to a variety of biotransformation products in human blood cells. Predominant metabolites were found to be conjugates of glutathione (GSH) isomers, namely M1-S-GSH and M1-N-GSH. Further sulfur-containing biotransformation products of M1 were conjugates with oxidized glutathione (M1-GSSG) and cysteine (M1-CYS) and the sulfated derivative of M1 (M1-sulfated). Other in vitro biotransformation products constituted the open-chained ester form of M1 (M1-COOH), hydroxybenzoic acid and the methylated (M1-methylated), acetylated (M1-acetylated), hydroxylated (M1-hydroxylated) and ethylated (M1-ethylated) derivatives of M1. Indeed, six of these in vitro metabolites, respectively M1-COOH, M1-sulfated, hydroxybenzoic acid, M1-S-GSH, M1-methylated and M1-acetylated, were also identified in vivo in blood cells of human volunteers after ingestion of Pycnogenol®. Related reference material was synthesized for reliable confirmation of the metabolites M1-GSH, M1-GSSG, M1-CYS and M1-COOH.
In the course of a randomized controlled clinical trial patients suffering from severe osteoarthritis ingested multiple doses of 200 mg/day Pycnogenol® for three weeks before they were scheduled for an elective knee replacement surgery. Various biological specimen, respectively blood cells, synovial fluid and serum samples, were to be analyzed to investigate the distribution and disposition of possibly bioactive constituents and metabolites. Therefore, highly sensitive methods were developed using liquid chromatography tandem mass spectrometry (LC-MS/MS)- technology because of the expected low concentrations of the analytes in the related matrices.
Initially, for each matrix different sample preparation techniques (protein precipitation, liquid-liquid extraction, solid phase extraction and useful combinations thereof) were compared to achieve maximum detection sensitivity of the analytes that were of highest interest, namely M1, ferulic acid and taxifolin. By comparing 32 various sample clean-up procedures in human serum, the highest recovery of the metabolite M1 was achieved using a liquid-liquid extraction with ethyl acetate and tert-butyl methyl ether at a serum pH-value of 3.2. A similar extraction method was also chosen for analyte detection in human synovial fluid after comparing 31 different sample preparation techniques. Whole blood or blood cells are difficult to handle because of their high viscosity and strong coloration. The QuEChERS (quick, easy, cheap, effective, rugged and safe) approach which was originally developed for the food safety and thus for the determination of pesticide residues in fruits and vegetables yielded the highest total recovery rate of M1 in human blood cells when assessing 18 different sample clean-up techniques. By applying the QuEChERS method for the first time for the simultaneous and highly sensitive quantification of selected polyphenols in human blood cells it was demonstrated that this fast and inexpensive technique can be applied in clinical fields for cleaning-up highly complex and thus challenging biological matrices. All developed methods for the different biological specimen were optimized to achieve maximum sensitivity of the target analytes. The determined lower limits of quantification (LLOQs) were sufficient for the quantification of the study samples. The LLOQs ranged from 113 pg/mL for taxifolin to 48 ng/mL for caffeic acid in blood cells and from 80 pg/mL for taxifolin to 3 ng/mL for caffeic acid in synovial fluid. In human serum the LLOQs even ranged down to 35 pg/mL for taxifolin and up to 8 ng/mL for caffeic acid. All analytical methods were subjected to a full validation according to current EMA and FDA guidelines and fulfilled those criteria, showing excellent performance and reliability of the developed and optimized methods.
Serum, blood cells and synovial fluid samples of the osteoarthritis patients were all processed with an enzymatic incubation with ß-glucuronidase/sulfatase to hydrolyse conjugates (phase-II-metabolism) prior the actual sample preparation. Additionally, serum samples of the osteoarthritis patients were prepared without enzymatic hydrolysis to determine the individual degree of conjugation with sulfate and glucuronic acid of the analytes.
All determined concentrations in the patients’ samples were in the lower ng/mL range. Notably, highest total concentrations of the polyphenols were not detected in serum, in which the degree of analyte conjugation with sulfate and glucuronic acid ranged from 54.29 ± 26.77% for catechin to 98.34 ± 4.40% for M1. The flavonoids catechin and taxifolin mainly partitioned into blood cells, whereas the metabolite M1, ferulic and caffeic acid primarily resided in the synovial fluid. The concentration of M1 in the blood cells was low, however, this could be explained by the previously observed extensive and rapid intracellular metabolism in vitro. This was now supported by the in vivo evidence in samples of patients who received Pycnogenol® in which the open-chained ester form of M1 (M1-COOH) as well as the glutathione conjugate of M1 (M1-GSH) were identified, indicating that M1 does not accumulate in its original form in vivo. Possibly, a variety of bioactive metabolites exist which might play an important role for the clinical effects of Pycnogenol®.
Although the study participants were requested to avoid polyphenol-rich food and beverages within the last two days before the blood samplings this was obviously difficult for most of the patients. Hence, no statistically significantly difference was observed in the mean polyphenol concentrations in serum, blood cells and synovial fluid between the intervention and the control group. Nevertheless, it was possible to identify marker compounds for Pycnogenol® intake under real life conditions with occasional or regular consumption of polyphenol-rich foods and beverages. Thereby, ferulic acid was found in serum samples exclusively after intake of Pycnogenol®, confirming that ferulic acid is a suitable marker of consumption of French maritime pine bark extract. Taxifolin was present in serum and synovial fluid exclusively in the intervention group indicating a role as further marker of Pycnogenol® intake. Taxifolin, ferulic acid and caffeic acid were detected in both serum and synovial fluid only in the intervention group. Moreover, the metabolite M1, taxifolin and ferulic acid were only detected simultaneously in all matrices (serum, blood cells and synovial fluid) after ingestion of Pycnogenol®.
Thus, deeper insights into the distribution of bioactive constituents and metabolites of Pycnogenol® into serum, blood cells and synovial fluid after oral administration to patients with severe osteoarthritis were gained. The present study provides the first evidence that polyphenols indeed distribute into the synovial fluid of patients with osteoarthritis where they might contribute to clinical effects.
Im Rahmen der Arbeit wurde eine Methode für die Quantifizierung von freiem 17β-Estradiol, Estron sowie der hydroxylierten und methylierten Metabolite im Brustgewebe entwickelt. Aufgrund der geringen Probengehalte erforderte dies eine gezielte Isolierung der Analyte aus der Probenmatrix sowie eine effektive Aufreinigung und Aufkonzentrierung, so dass eine Extraktion mit anschließender Festphasenextraktion durchgeführt wurde. Zudem wurde eine empfindliche Mess-Methode etabliert, welche auf Grundlage einer multi-reaction-monitoring-Methode, mittels Gaschromatographie und gekoppelten Triple-Quadrupol-Massenspektrometer, entwickelt wurde. Die Anwendbarkeit der Aufarbeitungs- und Mess-Methode wurde überprüft, indem diese auf 30 Realproben übertragen wurde. Dabei sind die ermittelten Gehalte mit den publizierten Daten der Gewebekonzentrationen von 17β-Estradiol, Estron und deren Metaboliten verglichen und Korrelationen mit ausgewählten Brustkrebs-begünstigenden Risikofaktoren betrachtet worden.
Um ein quantitatives Metabolitenprofil von 17β-Estradiol, Estron und deren Metaboliten im Gewebe zu erstellen, wurden mit Hilfe einer multi-reaction-monitoring-Methode für alle Metabolite ein spezifischer Quanti- und Qualifier-Übergang etabliert. Durch die Optimierung der Ionisierungs- und Kollisionsenergien sowie der Initial-, Transferline- und Ionenquell-Temperatur beziehungsweise der dwell-time wurden Methoden- und Geräte-bedingte Empfindlichkeitsverluste so weit wie möglich reduziert, so dass maximale Signalintensitäten aller Quantifier-Übergänge gewährleistet waren.
Zur gezielten Isolation sowie Aufreinigung und Anreicherung der Analyten,...
...so dass trotz der geringen Anzahl analysierter Gewebe-spenden der Einfluss des Body-Mass-Index und die Einnahme oraler Kontrazeptiva auf die Gehalte von 17β-Estradiol in der prämenopausalen Frau deutlich wurden.
Die entwickelte Mess-Methode ermöglicht den routinemäßigen Einsatz für die Quantifizierung von freiem 17β-Estradiol, Estron und deren Methyl-Catecholen in humanem Brustgewebe. Beim Vergleich der berechneten Nachweisgrenzen von Catechol-Estrogenen mit Literaturangaben wurde herausgestellt, dass empfindlichere flüssigchromatographische Methoden als Methode der Wahl bei deren Analytik heranzuziehen sind. Die Übertragung der in Standardlösungen durchgeführten Versuche zur enzymatischen Hydrolyse von Glucuronid-und Sulfat-Konjugaten auf Gewebematrix stellt für weiterführende Arbeiten den entscheidenden Ansatzpunkt dar, um ein quantitatives Metabolitenprofil von freiem und gebundenem 17β-Estradiol, Estron und den Metaboliten in Brustgewebe erstellen zu können.
Lattice forces are based on the attraction between the single moieties of molecules. The strength of lattice forces has an impact on the solid state and related physical properties such as melting point, boiling point, vapor pressure solvation and solubility. For solvation to occur, energy is required to break the lattice forces attracting ions and molecules among themselves. The energy for breaking up the attraction between the molecules is gained from the energy released when ions or molecules of the lattice associate with molecules of the solvent. Solubility is therefore, directly linked to the energy which is required to break the lattice forces and the energy which is liberated by solvation of the molecules or ions. Based on this relation, the lattice forces in two acidic compounds and a neutral compound were subsequently lowered by different approaches with the intention to increase the solubility, supersaturation, and dissolution rate.
The conversion to an ionic liquid and the embedding of the compound in a pH-sensitive matrix in an amorphous state were investigated with an acidic compound and its pro-drug. The tetrabutylphosphonium (TBPH) salt showed the most promising properties among the tested counter ions. It alters the properties of the compound from a highly crystalline physicochemical state to an amorphous readily soluble material showing supersaturation in a wider pH range and higher solubility than the sodium and potassium salts. A solid dispersion approach was developed in parallel. Solid dispersions with two different pH-sensitive polymers and different drug load were prepared by lyophilization to determine the miscibility of the compound and the polymer by differential scanning calorimetry (DSC). A miscibility of 50% of the amorphous acid with the pH-sensitive Eudragit L100-55 matrix and a miscibility of 40% with hydroxypropyl methylcellulose acetate succinate (HPMC-AS) was found. Both approaches, the TBPH salt and the solid dispersion based on the pH-sensitive Eudragit L100-55 were tested in vivo. The TBPH salt was dosed in a buffered solution to prevent precipitation in the acidic stomach pH. This resulted in BAV higher than the crystalline suspension but lower than the solid dispersion. There were no acute toxicology effects seen. Thus, TBPH was considered safe for further studies. The TBPH salts were very hygroscopic, sticky and prone to precipitation as free compound when exposed to low pH when simulating the passage through the stomach. Thus, the principle of the ionic liquid was combined with the principle of an amorphous solid dispersion. This mitigated the risk of precipitation of the TBPH salt during the passage of the stomach. Also delinquency upon open storage was improved by embedding the TBPH salt in a pH-sensitive polymer. Dissolution tests mimicking the pH gradient in the gastro intestinal tract confirmed the protective properties of the pH-sensitive polymer matrices against recrystallization at low stomach pH in vitro. Furthermore, supersaturation at pH ranges relevant in the intestines of preclinical species or humans was observed. The TBPH solid dispersion showed superior supersaturation behavior in vitro compared to the free acid in pH-sensitive matrix. However, equally increased bioavailability (BAV) was observed when the amorphous solid dispersion contained the free acid form or the TBPH salt. Absorption seemed to be so fast that the short in vitro supersaturation observed for the free from in pH-sensitive matrix was already sufficient for complete absorption within 15 - 30 minutes. This is in accordance with the short tmax of around 15 - 30 minutes after oral application of the low lattice force principles. The pharmacokinetic (PK) profile became the main focus of further optimization as the BAV was maximized already. Early maximal plasma concentration (tmax) went along with high maximal plasma concentration (Cmax) for the low lattice force principles. Central nervous system related side effects as consequence of the PK profile with such a high Cmax were likely to happen and therefore, the formulation principles were modified to maintain the doubled BAV and reduce the observed Cmax. Additionally, the compound showed a short half-life requiring a two times daily dose, which is suboptimal for a chronic treatment. The amorphous acid in pH-matrix showed a modified PK profile when dosed in a hydrogel but not in an oleo gel. Surprisingly, administration of the TBPH salt in pH-matrix suspended in oil showed a massive delay of the tmax to 8 hours and a reduction of Cmax by factor 2 - 3 with unchanged good BAV when administered as a suspension in oil without increased viscosity. TBPH salt solution with a high viscosity resulted in the same PK profile as when administered without increased viscosity.
The animal model was changed from rat to dog. The dose was limited to 15 mg/dog since they reacted much more sensitively to the drug. BAV at this dose level was 100% for the crystalline suspension already, thus the focus of this study was not increasing BAV but to achieve prolonged and/or delayed exposure using different formulation principles elaborated in rats before. An immediate release formulation of 3 mg was combined with a delayed/modified release principle containing 12 mg of the compound. An additional study arm was conducted with a remote controlled device programmed to deliver a first dose of 3 mg instantaneously after passing the stomach and a second dose of 12 mg when entering the caecum. The tmax remained short for all formulation principles and it seemed that delayed and modified release lead to BAV reduction. The modified PK profiles could not be translated to an oral dog model which endorsed the hypothesis of an absorption window; however, the in vitro results could be translated to a dog model for colonic absorption. A nanosuspension of the crystalline compound, the TBPH salt in pH-matrix and the TBPH salt of the pro-drug of the compound were administered rectally to determine colonic absorption. The nanosuspension showed exposure around the limit of quantification whereas the TBPH in pH-matrix showed 4% BAV and the pro-drug as TBPH salt in pH-matrix resulted in 12% BAV although the pro-drug is factor 3 less soluble. This was in line with the increased permeation of the pro-drug which was observed in the Caco2 experiments. The bioavailability was increased by using the low lattice force principles and validated the hypothesis for the acidic drug and its pro-drug in the colonic dog model. Chemical and physicochemical stability of the investigated solid dispersions was confirmed for at least 18 months at room temperature.
Amorphous solid dispersions were investigated to lower lattice forces of a neutral molecule. Solid dispersions are well known from literature; however, they are not frequently used as principles for dosage forms due to limitations in physical stability and complex manufacturing processes. A viable formulation principle was developed for a neutral compound assuming that the stability of a solid dispersion with a drug load below the maximal miscibility will be better than one which exceeds the maximal miscibility. The dispersed and amorphous state of the neutral compound resulted in a higher energy level and chemical potential compared to a crystalline form implying that they are thermodynamically instable and sensitive to recrystallization. This was confirmed by the fast recrystallization of an amorphous solid dispersion made from HPMC with 50% drug load which recrystallized within a few days. Solid dispersions with different drug loads in different polymers and in polymer mixtures were prepared by lyophilization. The miscibility of the compound and the polymer was determined by DSC as the miscibility is a surrogate for maximal stable drugload of the solid dispersion. HPMC was found to be miscible with 20% compound confirming the instability of the 50% HPMC solid dispersion observed earlier. Based on dosing needs, a miscibility/drug load of at least 30% was mandatory because of the dosing requirements to dose less than 1500 mg of final formulation. This was considered as maximal swallowable volume for later clinical development. Thus, all systems with a miscibility higher or equal to 30% drug in polymer were evaluated in an in vitro dissolution test and ranked in comparison with amorphous pure compound, crystalline compound and a 20% drug load solid dispersion made from HPMC. The HPMC based solid dispersion which gave good exposure in previous in vivo experiments did not support the high drugload that was needed. Therefore, similar in vitro behavior of this solid dispersion should result in similar in vivo performance. The polyvinylpyrrolidone (PVP) based solid dispersions scored with high drug load and medium initial kinetic solubility. The Soluplus based solid dispersion offer lower drug load and slightly lower initial kinetic solubility, but showed an extended supersaturation. The 4 best performing systems were evaluated in rats. They resulted in a short Tmax of 15 minutes and BAV higher than 85% indicating fast and complete absorption. The reference HPMC based solid dispersion with a drug load of 20% showed 65% BAV. This showed that higher drug loads were feasible and did not limit absorption in this animal model.
Since the estimated human dose required a higher formulation density than obtained from lyophilization or spray drying, melt extrusion of the solid dispersion was considered to be the most adequate technology. The process temperature needed to be below 200 °C as this value represents the degradation temperature of the polymers. It was investigated by differential scanning calorimetry whether the compound can be mixed with the molten polymer. None of the polymers could dissolve the crystalline compound below the degradation point of the polymer. The temperature had to be increased to 260 °C until the compound was molten together to a monophasic system with polymer. This resulted in degradation of the polymers. Therefore, different plasticizers and small organic molecules with similar functional groups as the compound were investigated on their ability to reduce the melting point of the mixture of polymer and compound. Positive results were obtained with several small molecules. Based on a literature review, nicotinamide had the least concerning pharmaceutical activities and was chosen for further development. Solid dispersions with the same composition as the ones tested in rat were prepared with 9% nicotinamide as softener. Extrusion without nicotinamide was not possible at 135 °C or at 170 °C whereas the addition of 9% nicotinamide led to a homogenous extrudate when processed at 135 °C. The solid state of the extrudates was not molecularly dispersed but the compound was in a crystalline state. They could not reach the in vitro performance observed for the lyophilized solid dispersions with Soluplus or PVP derivatives. Nevertheless, the performances in the supersaturation assay were comparable to the HPMC based lyophilized solid dispersion. The Soluplus and PVP based crystalline extrudates were evaluated in a dog PK showing that the crystalline solid dispersion does not enable BAV higher than 90% within 24 hours after application. In parallel, the hygroscopicity of the meltextrudates was investigated by DVS and the best performing system based on Kollidon VA64 was further optimized regarding the solid state after its extrusion. The minimal process temperature to obtain a fully amorphous solid dispersion was determined by hot stage X-ray powder diffraction analysis (XRPD) and confirmed by lab scale extrusion. Addition of 9% nicotinamide lowered the process temperature from 220 °C (without nicotinamide) to 200 °C with nicotinamide. The minimal temperature for obtaining crystal free material was independent of the nicotinamide amount as soon as it exceeded 9%. Lowering the process temperature with nicotinamide reduced the impurity levels from 3.5% at 220 °C to 1.1% at 200 °C. The fully amorphous extrudates performed now better in the in vitro supersaturation assay than the lyophilized amorphous HPMC solid dispersion and the crystalline extrudates which were extruded at 135 °C. The process was up-scaled to a pilot scale extruder with alternative screw designs increasing mechanical shear forces and mixing which enabled lower process temperatures. This resulted in a maximal process temperature of 195 °C when nicotinamide was present and 205 °C without nicotinamide. However, shorter process time and reduced process temperatures (compared to the lab scale equipment) resulted in impurity levels smaller than 0.5% for both compositions and temperatures and made the nicotinamide obsolete. The amorphous extrudates from the pilot scale extruder performed better in vitro than the crystalline extrudates from the lab scale extruder and the lyophilized HPMC solid dispersion. A comparable PK profile of the HPMC solid dispersion and the amorphous melt extruded formulation principle was anticipated from these in vitro results. This was confirmed by the pharmacokinetic profile in dogs after oral administration of the final extruded solid dispersion formulation which was equivalent with the pharmacokinetic profile of the HPMC based solid dispersion formulation. The assumption that using a drug load below the miscibility prevents the solid dispersion from recrystallization was verified at least for a limited time by a stability test at elevated temperatures for 3 months showing no change in solid state. This indicates the opportunities of the low lattice forces approach, but also showed the importance of developing principles first assuring stable solid state, performance in vitro and in vivo, tailor them in a second step based on performance and combine them with technology such as melt extrusion as third step. If these steps are done in the context of clinical needs and quality it can rationalize the development of a solid dispersion and minimalize the formulation related risks regarding biopharmacy and stability.
Protein-protein interactions play a crucial role in the development of drug delivery devices for the increasingly important biologicals, including antibodies, growth factors and cytokines. The understanding thereof might offer opportunities for tailoring carriers or drug proteins specifically for this purpose and thereby allow controlled delivery to a chosen target. The possible applications range from trigger-dependent release to sustained drug delivery and possibly permanently present stimuli, depending on the anticipated mechanism.
Silk fibroin (SF) is a biomaterial that is suitable as a carrier for protein drug delivery devices. It combines processability under mild conditions, good biocompatibility and stabilizing effects on incorporated proteins.
As SF is naturally produced by spiders and silkworms, the understanding of this process and its major factors might offer a blueprint for formulation scientists, interested in working with this biopolymer. The natural process of silk spinning covers a fascinating versatility of aggregate states, ranging from colloidal solutions through hydrogels to solid systems. The transition among these states is controlled by a carefully orchestrated process in vivo. Major players within the natural process include the control of spatial pH throughout passage of the silk dope, the composition and type of ions, and fluid flow mechanics within the duct, respectively. The function of these input parameters on the spinning process is reviewed before detailing their impact on the design and manufacture of silk based drug delivery systems (DDS). Examples are reported including the control of hydrogel formation during storage or significant parameters controlling precipitation in the presence of appropriate salts, respectively. The review details the use of silk fibroin to develop liquid, semiliquid or solid DDS with a focus on the control of SF crystallization, particle formation, and drug-SF interaction for tailored drug load.
Although we were able to show many examples for SF drug delivery applications and there are many publications about the loading of biologics to SF systems, the mechanism of interaction between both in solution was not yet extensively explored. This is why we made this the subject of our work, as it might allow for direct influence on pharmaceutical parameters, like aggregation and drug load.
In order to understand the underlying mechanism for the interaction between SF and positively charged model proteins, we used isothermal titration calorimetry for thermodynamic characterization. This was supported by hydrophobicity analysis and by colloidal characterization methods including static light scattering, nanoparticle tracking analysis and zeta potential measurements. We studied the effects of three Hofmeister salts – NaCl (neutral), NaSCN (chaotropic) and Na2SO4 (cosmotropic) – and the pH on the interaction of SF with the model proteins in dependence of the ratio from one to another. The salts impacted the SF structure by stabilizing (cosmotropic) or destabilizing (chaotropic) the SF micelles, resulting in completely abolished (cosmotropic) or strongly enhanced (chaotropic) interaction. These effects were responsible for different levels of loading and coacervation when varying type of salt and its concentration. Additionally, NaCl and NaSCN were able to prolong the stability of aqueous SF solution during storage at 25°C in a preliminary study.
Another approach to influence protein-protein interactions was followed by covalent modification. Interleukin-4 (IL-4) is a cytokine driving macrophages to M2 macrophages, which are known to provide anti-inflammatory effects. The possibility to regulate the polarization of macrophages to this state might be attractive for a variety of diseases, like atherosclerosis, in which macrophages are involved. As these cases demand a long-term treatment, this polarization was supposed to be maintained over time and we were planning to achieve this by keeping IL-4 permanently present in an immobilized way. In order to immobilize it, we genetically introduced an alkyne-carrying, artificial amino acid in the IL-4 sequence. This allowed access to a site-specific click reaction (Cu(I)-catalyzed Huisgen azide-alkyne cycloaddition) with an azide partner. This study was able to set the basis for the project by successful expression and purification of the IL-4 analogue and by proving the availability for the click reaction and maintained bioactivity. The other side of this project was the isolation of human monocytes and the polarization and characterization of human macrophages. The challenge here was that the majority of related research was based on murine macrophages which was not applicable to human cells and the successful work was so far limited to establishing the necessary methods.
In conclusion, we were able to show two different methods that allow the influence of protein-protein interactions and thereby the possible tailoring of drug loading. Although the results were very promising for both systems, their applicability in the development of drug delivery devices needs to be shown by further studies.
Ionische Flüssigkeiten (engl. Ionic Liquids = IL) sind organische Salze mit einem Schmelzpunkt von unter 100 °C und bieten einen interessanten Ansatz um die orale Bioverfügbarkeit von schlecht wasserlöslichen Arzneistoffen zu verbessern.
Aufgrund seiner schlechten Wasserlöslichkeit wurde aus dem Wirkstoff BGG492 der Novartis AG eine Ionische Flüssigkeit (IL) mit dem sterisch anspruchsvollen Gegenion Tetrabutylphosphonium hergestellt. Die IL ist ein amorpher, glasartiger Feststoff mit einem Schmelzpunkt von 57 °C. Die freie Säure (FS), das Kaliumsalz (BGG-K+) und die IL (siehe Abb. 69) wurden in festem Zustand mittels polarisationsmikroskopischen Aufnahmen, Röntgen-Pulverdiffraktometrie, Röntgenkristallstrukturanalysen, Infrarot-Spektroskopie und Festkörper-NMR-Spektroskopie untersucht.
Der ionische Charakter der IL in festem Zustand konnte mittels Bandenverschiebung der deprotonierten Sulfonamidgruppe im IR-Spektrum bestätigt werden. In der Röntgenkristallstrukturanalyse konnte gezeigt werden, dass sich die Moleküle der FS in Schichten anordneten, in denen jedes Molekül mit vier Nachbarmolekülen über Wasserstoffbrücken verbunden war. Das BGG-K+ kristallisierte als Monohydrat. In dieser Kristallstruktur bildeten die Kaliumkationen in der bc-Ebene mit den BGG-Anionen ober- und unterhalb Schichten. Im Gegensatz zu der FS waren keine intermolekularen Wasserstoffbrücken zu beobachten. Die 15N-Festkörper-NMR-Spektren des BGG-K+ und der IL zeigten die gleiche chemische Verschiebung für den unsubstituierten Stickstoffes N-1‘ der Pyrazolgruppe und belegten somit ebenfalls die ionische Struktur der IL im festen Zustand. Die amorphe Struktur der IL wurde mittels Röntgen-Pulverdiffraktometrie und Polarisationsmikroskop bestätigt und eine flüssigkristalline Phase konnte ausgeschlossen werden.
Die IL zeigte im Vergleich zu der FS eine 700-fach schnellere Auflösungsrate J und eine signifikante Verlängerung der Dauer der Übersättigung in wässriger Lösung. Der sprunghafte Anstieg der Kon-zentration in Lösung („spring“) und die Dauer der Übersättigung („parachute“) wurden mittels photometrischen und potentiometrischen Titrationen untersucht. Mit Hilfe der NMR-Spektroskopie konnte der Mechanismus der Übersättigung aufgeklärt werden. Das sterisch anspruchsvolle Gegenion Tetrabutylphosphonium verhinderte die Protonierung der deprotonierten Sulfonamidgruppe von BGG. In Lösung kam es zur Bildung von Aggregaten („Cluster“), in die sich das Gegenion teilweise einlagerte. Nach der Protonierung und der Bildung von Kristallisationskeimen präzipitierte die ungeladenen FS und der metastabile Zustand der Übersättigung („parachute“) brach zusammen.
Um den Einfluss der Struktur des Gegenions auf die Auflösungsrate und die Dauer der Übersättigung zu untersuchen, wurden ca. 40 Phosphonium- und Ammonium-Kationen synthetisiert. Die Schmelzpunkte der Phosphonium- und Ammonium-Salze wurden mittels dynamischer Differenzkalorimetrie (DSC) ermittelt. Für das Phosphonium-Salz P3332OH-Bromid konnte eine enantiotrope Umwandlung der Modifikationen mittels temperaturabhängiger XRPD-Messungen bestätigt werden. Die Zelltoxizitäts-Untersuchungen der Phosphonium- und Ammonium-Salze an humanen Leberzellen (HepG2), Nierenzellen (HEK 293T) und murinen Makro-phagenzellen (J774.1) zeigten, dass mit höherer Lipophilie die Zelltoxizität zunahm. Polare Kationen zeigten keine Zytotoxizität (IC50 > 1000 µM). Die Zelltoxizität der Ammonium-Salze war im direkten Vergleich mit den Phosphonium-Salzen etwas geringer.
Die synthetisierten Phosphonium- und Ammonium-Salze, die als Chloride-, Bromide- und Iodide vorlagen, wurden durch Anionenaustausch in Hydroxide umgewandelt. Die Ionischen Flüssigkeiten wurden in einer Säure-Base-Reaktion mit der freien Säure des BGG-Moleküls und den Hydroxiden hergestellt. Der ionische Charakter konnte mittels Bandenverschiebung der deprotonierten Sulfonamidgruppe im IR-Spektrum bestätigt werden.
Die Substanzen waren amorph (XRPD) und die Glasübergangstemperaturen (DSC) bewegten sich für die Mono-Kationen im Bereich zwischen 40 °C – 97 °C, für Dikationen 81 °C - 124 °C und für Trikationen 124 °C - 148 °C. Damit erfüllten einige Substanzen die Definition einer Ionischen Flüssigkeit nicht (Smp. < 100 °C) und wurden daher als Niedrig-Gitter-Enthalpie-Salze (low lattice enthalpy salt = LLES) bezeichnet. Die ILs und LLES zeigten signifikante Unterschiede in der Auflösungsrate J, der Übersättigungszeit und der Wasserdampfsorption.
In dieser Arbeit konnte gezeigt werden, dass allein durch die Auswahl des Gegenions wichtige Parameter für die orale Bioverfügbarkeit gesteuert werden können. Durch diesen Ansatz war es möglich, aus dem sehr schlecht wasserlöslichen Arzneistoff BGG492 Ionische Flüssigkeiten bzw. LLES herzustellen, die sich drastisch schneller auflösten und teilweise über mehrere Stunden übersättigte Lösungen bildeten. Insgesamt zeigte sich, dass durch eine Zunahme der Polarität des Gegenions eine größere Auflösungsrate J und eine geringere Zelltoxizität erzielt werden konnten. Jedoch verringerte sich dadurch die Dauer der Übersättigung in Lösung und erhöhte die Hygroskopizität der ILs und LLES.