"In vitro" and "in vivo" drug targeting using biotinylated immunoliposomes

The aim of my thesis was the optimization, characterization and application of a new, innovative drug carrier system which can be used to target pharmaceuticals to diseased tissues and organs. In the present study, a non-covalent biotin-streptavidin coupling procedure for the preparation of pegylated immunoliposomes is presented which simplifies the attachment of targeting vectors to sterically stabilized liposomes. The OX26 monoclonal antibody against the rat transferrin receptor was used as a targeting vector. Immunostaining experiments with the OX26 monoclonal antibody followed by fluorescent confocal microscopy revealed immunofluorescence labeling of the transferrin receptor on muscle and on glioma cells. Uptake experiments with these cells demonstrated cellular uptake and accumulation of small molecules (daunomycin, fluorescent probes) or macromolecules such as fluorescent oligonucleotides, within an intracellular compartment. Cellular uptake of liposomal daunomycin by multidrug-resistant cells was dose- and time-dependent and was associated with a clear pharmacological (i.e. cytotoxic) effect. Cytotoxic effects of liposomal formulations of daunomycin, in contrast to the free drug, were apparent only after prolonged incubation periods being indicative of a slow intracellular unpacking and release of liposomal daunomycin. With respect to expression plasmids (i.e. gene targeting), only marginal levels of gene expression were observed in vitro. Pharmacokinetics and tissue distribution studies in the rat revealed a substantially increased plasma half-live of liposomal drug compared to free drug. Brain accumulation of daunomycin in OX26-immunoliposomes occurred in higher levels as compared to brain uptake of free daunomycin or daunomycin incorporated within pegylated liposomes or unspecific IgG2a isotype control immunoliposomes. Such OX26-mediated effects were not observed in other tissues such as spleen, liver, muscle or kidney. Our experiments indicate that immunoliposomes can be used to target small drug molecules as well as macromolecules, such as oligonucleotides and expression plasmids, to cells and tissues. These findings demonstrate that immunoliposomes are a versatile and promising tool for future drug development in regard to both, galenic and therapeutical research. For clinical administrations, a better understanding of cellular uptake and release mechanisms are needed

Strategies for Accelerating the Development of Catalytic Enantioselective Reactions

The development of enantioselective catalytic processes for the manufacture of chiral intermediates is a very complex endeavor and can be very time consuming and expensive. In this contribution the major issues which might lead to long development times will be discussed and strategies to deal with these problems are described. The general part is illustrated with the approach Solvias has chosen for assisting and supporting the development of enantioselective homogeneous hydrogenation processes, at the moment the most important industrial application of asymmetric catalysis. Special emphasis is given to the application of high-throughput screening (HTS) using a Symyx HiP system and the description of the Solvias portfolio of chiral ligands which makes a broad variety of diphosphine ligands available for all phases of process development from the first screening experiments to the large-scale manufacturing phase. Four case histories serve to illustrate the generic description of the development process

A review of the transportability of cognitive therapy for the treatment of PTSD among South African rape survivors

This study aimed to evaluate the transportability of cognitive therapy (CT) for rape survivors with PTSD to South African conditions. Ten local treatment outcome studies investigating the transportability of CT were identified and appraised. The common elements of CT for PTSD including psychoeducation, exposure therapy and cognitive restructuring of trauma-related appraisals were found to be transportable to local contexts. Contextual factors that can complicate treatment delivery were also identified namely exposure to multiple traumatic events, HIV, absence of safety and support in the external environment and language barriers. The augmentations made to an existing evidence-based treatment protocol to address these contextual factors are described.DHE

Targeting of daunomycin using biotinylated immunoliposomes: Pharmacokinetics, tissue distribution and in vitro pharmacological effects

Biotinylated immunoliposomes were prepared by a non-covalent (biotin-streptavidin) coupling procedure and conjugated to the OX26 monoclonal antibody directed against the rat transferrin receptor. In vitro, these biotinylated immunoliposomes were used to by-pass P-glycoprotein in multidrug-resistant RBE4 brain capillary endothelial cells and thereby to achieve 2- to 3-fold higher intracellular accumulation of liposomal daunomycin as compared to free drug. The extent of cellular uptake of liposomal daunomycin was dose- and time-dependent, was inhibited by competition with unbound OX26 and was associated with a pharmacological (i.e. cytotoxic) effect. Cytotoxic effects of liposomal formulations of daunomycin, in contrast to the free drug, were apparent only after prolonged incubation periods being indicative of a slow intracellular unpacking and release of liposomal daunomycin. Pharmacokinetics and tissue distribution studies in the rat revealed brain accumulation of daunomycin in OX26-immunoliposomes to higher levels as compared to brain uptake of free daunomycin, or daunomycin incorporated within pegylated liposomes or within unspecific IgG(2a) isotype control immunoliposomes. Such OX26-mediated effects were not observed in other tissues such as spleen, liver, muscle or kidney

Targeting of skeletal muscle in vitro using biotinylated immunoliposomes.

In the present study, a non-covalent (biotin-streptavidin) coupling procedure for the preparation of pegylated immunoliposomes is presented, which simplifies the attachment of targeting vectors to sterically stabilized liposomes. A biotinylated poly(ethylene glycol) (PEG)-phospholipid [bio-PEG-distearoylphosphatidylethanolamine (DSPE)] was used as a linker between a streptavidin-conjugated monoclonal antibody (mAb) (i.e. the OX26 mAb raised against the rat transferrin receptor) and 150 nm liposomes. OX26-streptavidin had a biotin binding capacity of two to three biotin molecules per OX26-streptavidin conjugate. Immunostaining experiments with the OX26 mAb followed by fluorescent confocal microscopy revealed immunofluorescence labelling of the transferrin receptor on skeletal muscle, as well as in L6 cells, a continuous cell line derived from rat skeletal muscle. Uptake experiments with L6 cells using the OX26 mAb, fluorescence-labelled OX26-streptavidin or fluorescent OX26-immunoliposomes demonstrated cellular uptake and accumulation within an intracellular compartment of the OX26 mAb and its conjugates. Cellular uptake of OX26 conjugates was sensitive to competition with free OX26 antibody. In summary, these studies describe the design of biotinylated immunoliposomes as a universal drug transport vector and their potential for targeting of the transferrin receptor of skeletal muscle