Imiquimod based transcutaneous immunization – insights and novel concepts

This work aims at developing a transcutaneous immunization (TCI) approach in order to activate cytotoxic T-cells. A tumor specific immune response was therefore generated by the TLR7-Agonist imiquimod. Five commercially available creams including the innovators product Aldara® 5% creme were assessed to ascertain their capability to induce an immune response in C57BL/6 mice after dermal administration. Moreover, creams were investigated regarding their imiquimod permeation in a Franz-diffusion cell model. Results obtained from this study were used to develop novel formulation approaches based on dissolved state imiquimod in a submicron scale range. High pressure homogenization ensured emulsification as well as particle size reduction. A freeze dried spreadable solid nanoemulsion based on sucrose fatty acid esters and oil components represented a major formulation approach. Within the scope of this approach the influence of pharmaceutical oils i.e. middle chain triglycerides, avocado oil, jojoba wax, and squalen was assessed towards their TCI performance. Furthermore, an aqueous jojoba wax based emulsion gel was developed. Unlike the innovators product, all formulations demonstrated a distinctly reduced imiquimod permeation across murine skin, a fact particularly evident in case of jojoba wax. Squalen significantly augmented in vivo immune response (p≤0.05 Mann-Whitney-Test). The emulsion gel demonstrated a 10fold decrease of imiquimod permeation. In comparison with the innovators product, the emulsion gel induced an equal immune response with a simultaneously enhanced tumor rejection in a mouse model.

Transkutane Immunisierung - Mechanismen und Optimierungsstrategien

In dieser Arbeit wurde ein etabliertes Immunisierungsmodell auf der Basis eines synthetischen TLR 7-Liganden zur Aufhebung der suppressiven Eigenschaften von UV B-Strahlung verwendet. Es konnte gezeigt werden, dass nach Ermittlung eines geeigneten Immunisierungsprotokolls mit UV B die durch TCI vermittelte ausschließlich primäre Immunantwort verstärkt werden konnte, so dass eine Gedächtnisantwort induziert werden konnte. Durch die Immunisierung verschiedenster knockout sowie transgener Stämme konnte bewiesen werden, dass die Reaktion auch nach zusätzlicher UV B-Bestrahlung abhängig von TLR 7 und dem Adaptormolekül MyD88 ist. Bei der Aufklärung der zellulären Mechanismen, die dieser Immunisierungsmethode zu Grunde liegen, konnten dermale DCs, sowie GR1+-Zellen als wichtige Mediatoren identifiziert werden. Eine wesentliche Funktion von epidermalen Langerhans Zellen konnte in diesem Zusammenhang ausgeschlossen werden. Des Weiteren konnte gezeigt werden, dass auf Zytokinebene Typ I Interferone eine tragende Rolle spielen und die Produktion von IL-12p35 in den Haut-drainierenden Lymphknoten angeregt wird. Supprimiert wird die Immunantwort durch regulatorische T-Zellen, sowie durch die Freisetzung von IL-10, welches nicht ausschließlich von regulatorischen T-Zellen produziert wird. Die Applikation eines blockierenden IL-10-Rezeptor Antikörpers verhinderte die IL-10-vermittelte Suppression und führte zu einer weiteren Verstärkung der Immunantwort. Durch die induzierte Gedächtnisantwort nach UV B/TCI konnte das Immunisierungs-Modell in einem therapeutischen Tumormodell angewandt werden und führt hier zu einer verstärkten Abstoßung von Tumoren, verglichen mit TCI alleine, was die Basis für zukünftige nadelfreie Vakzinierungen für die Behandlung von Krebs oder persistierender Infektionen darstellen könnte.

Der Einfluss der T-Zellavidität und der TLR-vermittelten Aktivierung Dendritischer Zellen der Haut auf die Funktion zytotoxischer T-Zellen

Zytotoxische T-Zellen (CTL) vermitteln die effektive und gezielte Lyse Virus-infizierter und maligner Zellen. Um den Einfluss der T-Zellavidität auf die Immunodominanz von CTL-Antworten zu untersuchen, wurde ein neues Mausmodell mit zwei T-Zellrezeptor (TCR)-transgenen Stämmen etabliert. Die T-Zellen der beiden Stämme exprimieren denselben TCR in unterschiedlicher Dichte. Studien mit diesem Modell zeigten, dass bei identischen Vorläuferfrequenzen die hochaviden T-Zellen nach Immunisierung auf Kosten der niederaviden T-Zellen proliferieren. Sind die niederaviden T-Zellen jedoch im Überschuss vorhanden, so können sie die Antwort dominieren. Hieraus lässt sich schließen, dass die CTL Immunodominanz durch die Avidität und die Vorläuferfrequenz der T-Zellklone bestimmt wird. Darüber hinaus wurde ein neues transkutanes Immunisierungsverfahren (TCI) entwickelt. Die Applikation eines CTL Epitops zusammen mit dem synthetischen Toll-like Rezeptor 7 Liganden Imiquimod führt zur effizienten Aktivierung TCR-transgener CTLs. Eine in Wild-typ Mäusen generierte CTL-Antwort gegen das SIINFEKL Epitop aus dem Ovalbumin vermittelt die effiziente Lyse inokulierter Ovalbumin-transgener Thymomzellen. Schließlich konnte gezeigt werden, dass regulatorische T-Zellen die durch TCI vermittelte CTL Aktivierung inhibieren.

Design and physicochemical characterisation of a bioadhesive patch for dose-controlled topical delivery of imiquimod

Clinical use of the imidazoquinoline immunomodulator imiquimod for the topical treatment of dysplastic and neoplastic lesions has increased markedly in recent years. However, despite guidance from the manufacturer of the proprietary imiquimod cream, there seems to be little consensus between clinicians as to the topically applied dose. Given that patients often apply the cream themselves at home, further dosing variability is expected and, consequently, accurate comparison of the results of different published studies is dif?cult. This paper describes, for the ?rst time, the formulation and physicochemical characterisation of a bioadhesive patch for dose-controlled topical delivery of imiquimod as well as a new HPLC method for sensitive ?uorescence determination of imiquimod released from such systems. Patches containing imiquimod loadings of 4.75, 9.50 and 12.50 mg cm-2 all released signi?cantly more drug across a model membrane than the proprietary cream over a period of 6 h. Inclusion of imiquimod in patches did not adversely affect their physicochemical properties. Of major importance, patches contained de?ned drug loadings per unit area; therefore, their use could reduce inter-clinician variability. This would make critical comparison of clinical studies and determination of an appropriate imiquimod dose for successful treatment much simpler. Since bioadhesive formulations are capable of adhering to body tissues in moist environments, the use of a bioadhesive patch system may allow extension of the clinical uses of imiquimod to the treatment of neoplastic conditions of the oral cavity and cervix, as well as the vulva. © 2005 Elsevier B.V. All rights reserved.

Molecularly imprinted polymers for the extraction of imiquimod from biological samples using a template analogue strategy

Molecularly Imprinted Polymers (MIPs) against imiquimod, a highly potent immune response modifier used in the treatment of skin cancer, were synthesised using a template analogue strategy and were compared with imprints of the drug itself. An investigation of the complexation between the functional monomer and the template analogue revealed an association constant of 1,376 ± 122 M-1, significantly higher than previously reported values for similar systems. The binding characteristics of the synthesised imprinted polymers were evaluated and extremely strong binding for imiquimod was observed while imprinting factors as high as 17 were calculated. When applied as sorbents in solid-phase extraction of imiquimod from aqueous, urine and blood serum samples, clean extracts and recoveries up to 95% were achieved, and it is concluded that while imiquimod imprints exhibited higher capacity for the drug, template analogue imprints are more selective. The results obtained suggest potential applications of imiquimod imprints as sorbents in rapid extraction and monitoring of undesirable systemic release of the drug.

Le bouquet présenté au Roy, le jour de sa feste, par le sieur J. B. D. L. R.

[Mazarinade. 1649]

Lettre du Roy, envoyée à Mgr l'abbé de Sainct Germain Desprez, ou à son vicaire général en son absence, pour l'exhorter de faire chommer la feste de sainct Joseph dans l'estendüe de sa jurisdiction

[Acte royal. 1661-03-16. Paris]

La seconde suite des triolets royaux sur tout ce qui s'est passé à Paris de plus remarquable depuis la feste de Sainct Louys...

[Mazarinade. 1649]

An artificial nanoemulsion carrying paclitaxel decreases the transplant heart vascular disease: A study in a rabbit graft model

Objective: In previous studies cholesterol-rich nanoemulsions (LDE) resembling low-density lipoprotein were shown to concentrate in atherosclerotic lesions of rabbits. Lesions were pronouncedly reduced by treatment with paclitaxel associated with LDE. This study aimed to test the hypothesis of whether LDE-paclitaxel is able to concentrate in grafted hearts of rabbits and to ameliorate coronary allograft vasculopathy after the transplantation procedure. Methods: Twenty-one New Zealand rabbits fed 0.5% cholesterol were submitted to heterotopic heart transplantation at the cervical position. All rabbits undergoing transplantation were treated with cyclosporin A (10 mg . kg(-1) . d(-1) by mouth). Eleven rabbits were treated with LDE-paclitaxel (4 mg/kg body weight paclitaxel per week administered intravenously for 6 weeks), and 10 control rabbits were treated with 3 mL/wk intravenous saline. Four control animals were injected with LDE labeled with [(14)C]-cholesteryl oleate ether to determine tissue uptake. Results: Radioactive LDE uptake by grafts was 4-fold that of native hearts. In both groups the coronary arteries of native hearts showed no stenosis, but treatment with LDE-paclitaxel reduced the degree of stenosis in grafted hearts by 50%. The arterial luminal area in grafts of the treated group was 3-fold larger than in control animals. LDE-paclitaxel treatment resulted in a 7-fold reduction of macrophage infiltration. In grafted hearts LDE-paclitaxel treatment reduced the width of the intimal layer and inhibited the destruction of the medial layer. No toxicity was observed in rabbits receiving LDE-paclitaxel treatment. Conclusions: LDE-paclitaxel improved posttransplantation injury to the grafted heart. The novel therapeutic approach for heart transplantation management validated here is thus a promising strategy to be explored in future clinical studies. (J Thorac Cardiovasc Surg 2011;141:1522-8)

Photodynamic inactivation of biofilms formed by Candida spp., Trichosporon mucoides, and Kodamaea ohmeri by cationic nanoemulsion of zinc 2,9,16,23-tetrakis(phenylthio)-29H, 31H-phthalocyanine (ZnPc)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Antimicrobial and physical-mechanical properties of pectin/papaya puree/cinnamaldehyde nanoemulsion edible composite films

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Development, characterization, and in vitro trials of chloroaluminum phthalocyanine-magnetic nanoemulsion to hyperthermia and photodynamic therapies on glioblastoma as a biological model

A glioblastoma multiforme (GBM) is the highest grade glioma tumor (grade IV) and is the most malignant form of astrocytomas. Grade IV tumors, which are the most malignant and aggressive, affect people between the ages of 45 and 70 years. A GBM exhibits remarkable characteristics that include excessive proliferation, necrosis, genetic instability, and chemoresistance. Because of these characteristics, GBMs are difficult to treat and have a poor prognosis with a median survival of less than one year. New methods to achieve widespread distribution of therapeutic agents across infiltrative gliomas significantly improve brain tumor therapy. Photodynamic therapy (PDT) and hyperthermia (HPT) are well-established tumor therapies with minimal side effects while acting synergistically. This study introduces a new promising nanocarrier for the synergistic application of PDT and magnetic hyperthermia therapy against human glioma cell line T98 G, with cellular viability reduction down to as low as 17% compared with the control. (C) 2012 American Institute of Physics. [doi:10.1063/1.3671775]