Da die Arzneistoffpermeation durch okulare Gewebe einen entscheidenden Einfluss auf die Heilung vieler Augenleiden hat, wurde die in vitro-Permeation hydrophiler und lipophiler Arzneistoffe durch okulare Gewebe und Zellkulturen in dieser Arbeit untersucht. Die Dissertation befasst sich vorrangig mit der Permeation des hydrophilen Modellarzneistoffs Pilocarpinhydrochlorid (P-HCl) durch isolierte Schweinecornea (SC), Schweinesklera, Kaninchenkonjunktiva und corneale bzw. konjunktivale Kaninchenepithelzellkultur. Der Einfluss verschiedener Formulierungsparameter wie Benzalkoniumchlorid (BAC), Natriumedetat, pH-Wert und Tonizität auf die P-HCl-Permeation wurde untersucht. Dabei konnte eine gute Korrelation zwischen isolierten Geweben und Zellkulturen in Reaktion auf die Variation der Formulierungsparameter festgestellt werden. Unter den getesteten Parametern zeigte BAC den größten Enhancereffekt. Weiterhin wurden vergleichende Permeationsstudien an gelaserter SC mit P-HCl und dem relativ lipophilen Diclofenac-Natrium (D-Na) durchgeführt. Das Entfernen von Epithelschichten der SC mittels Excimer-Laser sollte den Heilungsverlauf, vor allem aber die zeitabhängige Reepithelisierung der Cornea nach erfolgter Photorefraktiver Keratektomie, simulieren. Die Ergebnisse dieser Studie zeigten, dass unterschiedliche Epitheldicken einen signifikanten Einfluss auf die P-HCl-Permeation haben. Im Gegensatz dazu blieb die D-Na-Permeation nahezu unbeeinflusst. Ein weiteres Anliegen dieser Arbeit bestand darin, eine okular applizierbare Formulierung des Immunsuppressivums Mycophenolatmofetil (MMF) zu entwickeln. Sowohl für das Prodrug MMF als auch für dessen aktiven Metaboliten Mycophenolsäure (MPA) wurde die Permeabilität von SC getestet. Ausgewählt wurde eine Zubereitung, die 1% MMF in Glutathion-Bicarbonat-Ringerlösung enthält und mit 10% Hydroxypropyl-beta-Cyclodextrin versetzt ist. Diese Suspension wurde bei 121° C und 200 kPa für 15 min autoklaviert, um das schwerlösliche MMF in Lösung zu bringen. Da der Ester MMF bei der Herstellung der Testlösung einer teilweisen Hydrolyse zu MPA unterliegt, außerdem eine minimale in vitro-Verfügbarkeit aufweist und sehr schwer wasserlöslich ist, sollte MPA in Augentropfenformulierungen der Vorzug gegeben werden. Die Korrelierbarkeit mit in vivo-Resultaten ist jedoch im Rahmen dieser Arbeit nicht untersucht worden, so dass die Ergebnisse als Grundlage für Permeationsstudien am in vivo-Modell zu bewerten sind. / The permeation of drugs through ocular tissues plays an important role in healing of various eye diseases. The objective of this work was to investigate the in vitro permeability of hydrophilic and lipophilic drugs through ocular tissues and cell cultures. Mainly, the permeability of the hydrophilic model drug pilocarpine hydrochloride (P-HCl) through isolated pig cornea (PCr) and sclera, rabbit conjunctiva, and rabbit conjunctival or corneal epithelial cell culture was compared. Additionally, the study included investigations about the influence of the formulation parameters benzalkonium chloride (BAC), ethylene diamine tetra acetic acid disodium salt, pH value and tonicity on the permeability of the small drug. In summary, a good correlation between the isolated tissues and cell cultures with regard to P-HCl transport could be observed. In general, BAC caused the most facilitated drug transport within the formulation parameters. Furthermore, the permeation of P-HCl and the lipophilic diclofenac-sodium (D-Na) through lasered PCr was studied. To investigate the effects of photorefractive keratectomy on drug permeation, excimer laser ablations with varying depths were performed on isolated pig eyes. As a result, P-HCl demonstrated a significant enhancement of permeation in relation to the ablation depth. In contrast, corneal epithelial thickness scarcely influenced the permeation rate of D-Na. Another aim of this work was to develop an appropriate application form for topical ocular use of the immunomodulating substance mycophenolate mofetil (MMF) and to investigate the in vitro permeability of PCr for the prodrug MMF and its parent substance mycophenolic acid (MPA). The test formulation consisted of Glutathion-bicarbonate-Ringer's solution, 10% hydroxypropyl- beta-cyclodextrin and 1% MMF. To reach a concentration of 1% of the poor soluble MMF a treatment under autoclaving conditions at 121° C and 200 kPa for 15 min was needed. MPA should be preferred in eye drops because of an higher in vitro availability compared to MMF, an hydrolysis of MMF to MPA in the cornea during permeation and the poor water solubility of MMF. The correlation with in vivo results was not studied in this work but the findings can be assumed as basis for investigations on in vivo models.

Die Verwendung von Cyclodextrinen (CDen) als Hilfsstoffe zur topischen Anwendung in der Ophthalmologie hat in den letzten Jahren zunehmend an Bedeutung gewonnen. Hierbei steht die Verbesserung der Wasserlöslichkeit schwerlöslicher Arzneistoffe, die Modifizierung der Bioverfügbarkeit sowie die Verminderung okularer Reizungen im Vordergrund. Diese Arbeit untersuchte die corneale In-vitro-Permeation von Betarezeptorenblockern (partiell mit Pilocarpin kombiniert) im Zusammenhang mit alpha-CD und den hydroxypropylierten (HP) alpha-, beta- bzw. gamma-CD-Derivaten. Der Zusatz von CDen führte teilweise zu einer signifikanten Veränderung der Permeationsparameter, verglichen mit denjenigen der CDfreien Lösungen. Weiterhin wurden das schwerlösliche Prodrug Mycophenolatmofetil (MMF) und die aktive Wirksubstanz Mycophenolsäure (MPA) mit HP-beta-CD in Lösung gebracht und anschließend die Wirkstoffverteilung im Kaninchenauge in vivo bestimmt. Im Vergleich zu einer MMF-Suspension waren die MPA-Spiegel in Cornea und Kammerwasser nach 60 min für die HP-beta-CD-haltige MMF-Lösung deutlich erhöht. Ferner wurden für die neuen, extrem schwer wasserlöslichen Selektiven Glucocorticoid-rezeptoragonisten (SEGRA) - ZK 216771 und ZK 247756 - innovative Augentropfen-formulierungen entwickelt. So wurde ZK 216771 mit Ethanol und HP-gamma-CD in Lösung gebracht und für ZK 247756 eine HP-gamma-CDhaltige Mikroemulsion entwickelt. In beiden Fällen war ein spezielles Herstellungsverfahren erforderlich. ZK 247756 konnte auf diese Weise nach Keratoplastik bei Ratten erfolgreich angewendet werden (signifikante Verlängerung der Transplantatüberlebenszeit im Vergleich zu einer unbehandelten Kontrollgruppe bzw. Vehikelgruppe). / In the past years, cyclodextrins (CDs) became more and more an important excipient for drug formulations. The main use of CDs in ophthalmic drug delivery is to increase the water solubility of lipophilic drugs, to modify drug bioavailability and to reduce ocular irritation. In the present study, the corneal permeability of different betablockers (in part combined with pilocarpine) in the presence of alpha-CD and hydroxypropylated (HP) alpha-, beta- and gamma-CD was examined. Addition of CDs often led to significant changes in permeation parameters, compared to CD-free solutions. The prodrug mycophenolate mofetil and its active drug mycophenolic acid (MPA) were solubilized by HP-beta-CD. The intraocular availability of these solutions was studied in the rabbit eye in vivo in comparison to a MMF-suspension. Applying MMF/HP-beta-CD-solution, the MPA level in cornea and aqueous humour (after 60 min) turned out to be significant higher than those of the MMF-suspension. In addition, for the extremely low soluble selective glucocorticoidreceptor agonists (SEGRA) - ZK 216771 and ZK 247756 - innovative drug formulations were developed. ZK 216771 was solubilized with ethanol and HP-gamma-CD in buffer. A microemulsion, containing also HP-gamma-CD, served as vehicle for ZK 247756. For both formulations, a special manufacturing process was required. In this way, ZK 247756 could be applied in rabbits after corneal transplantation. Topical application of this formulation was highly effective in prolonging the mean survival time of corneal grafts as compared to untreated controls or animals that received the vehicle only.

Introduction:Uveitis in an ocular inflammation affecting mostly people of working age. Uveitis is responsible for severe visual impairment despite of expanding new therapeutics. The animal models of uveitis were established, because the wide clinical variability of uveitis limits the studies in human medicine. The goal our project was to establish a reproducible model of experimental autoimmune uveitis in Czech Republic, and further on this model to observe the frequency of CD3+ and F4/80+ cells in retina, to assess the influence of microbial environment on intensity of intraocular inflammation and to test the therapeutical possibilities. Material and methods: The C57BL/6J mice were immunized by retinal antigen (IRBP 1-20, interphotoreceptor retinoid binding protein), enhanced by complete Freund's adjuvant and pertussis toxin and mild posterior autoimmune uveitis was induced. The mice were bred in conventional and germ-free (gnotobiotic) conditions. The uveitis intensity was evaluated in vivo biomicroscopically and post mortem histologically on hematoxylin eosin stained sections according to the standard protocol. The histological eye specimen were analyzed also by imunohistochemisty and by flow cytometry. Each experiment was performed for 35 days. The conventional mice with uveitis were treated...

The aim of this work was to gain new knowledge about mechanisms of autoimmune uveitis and to test new therapeutic possibilities that have not yet been studied in uveitis or whose effect is questionable. The main emphasis was placed on the role of microorganisms in the process of uveitis. A mouse model of experimental autoimmune uveitis including a germ-free model was used to achieve the aims and samples of patients' intraocular fluids were analyzed. In the experimental model, the intensity of inflammation was evaluated in vivo clinically and post mortem histologically. The effect of immunomodulatory treatment was evaluated. The intensity of inflammation was compared between groups of germ-free and conventional mice. The therapeutic effect of antibiotics administered to affect microbiome was investigated in conventional mice. In intraocular fluid samples of patients with autoimmune uveitis signs of infection were monitored and levels of cytokines and other factors were evaluated. Evaluation of the effect of immunomodulatory therapy has demonstrated the efficacy of mycophenolate mofetil, which supports its wider use in the treatment of autoimmune posterior uveitis in human medicine. The decrease in bacterial load has led to a decrease in the intensity of inflammation, thereby confirming the importance of...

Introduction:Uveitis in an ocular inflammation affecting mostly people of working age. Uveitis is responsible for severe visual impairment despite of expanding new therapeutics. The animal models of uveitis were established, because the wide clinical variability of uveitis limits the studies in human medicine. The goal our project was to establish a reproducible model of experimental autoimmune uveitis in Czech Republic, and further on this model to observe the frequency of CD3+ and F4/80+ cells in retina, to assess the influence of microbial environment on intensity of intraocular inflammation and to test the therapeutical possibilities. Material and methods: The C57BL/6J mice were immunized by retinal antigen (IRBP 1-20, interphotoreceptor retinoid binding protein), enhanced by complete Freund's adjuvant and pertussis toxin and mild posterior autoimmune uveitis was induced. The mice were bred in conventional and germ-free (gnotobiotic) conditions. The uveitis intensity was evaluated in vivo biomicroscopically and post mortem histologically on hematoxylin eosin stained sections according to the standard protocol. The histological eye specimen were analyzed also by imunohistochemisty and by flow cytometry. Each experiment was performed for 35 days. The conventional mice with uveitis were treated...

Targeting mycophenolate mofetil for graft-versus-host disease prophylaxis after allogenic blood stem cell transplantation

Targeting mycophenolate mofetil for graft-versus-host disease prophylaxis after allogenic blood stem cell transplantation:II Deckblatt III Gutachterblatt IV Widmung V Inhaltsverzeichnis VI.1 Publikation VI.2 Original-Artikel VII Abkürzungsverzeichnis VII.1 Selbständigkeitserklärung VII.2 Erklärung über den Eigenanteil bei Erstellung des Manuskriptes VII.3 Lebenslauf VII.4 Danksagung VII.5 Thesen