Peptide and drug interactions in murine models : in vitro and in vivo studies with microphysiometry, confocal microscopy and functional magnetic resonance imaging

Abstract

The present thesis describes in three parts how living cells can be investigated on different biological levels, and how these techniques can be used to non-invasively evaluate the effect of drugs in the context of biomedical and pharmaceutical research. In the first part, Pgp activation is studied in living cells studied in culture. Here, microphysiometry, monolayer techniques and computer modeling were applied to investigate the interaction of drugs with human P-glycoprotein (Pgp). Pgp is a transmembrane ABCtransporter that protects cells from a wide variety of toxic substances. Pgp is helpful under most biological conditions since it provides natural protection from toxins, but its activity can lead to resistance development in chemotherapy e.g. of cancers and microbes. In particular, the reported techniques provided a novel method to study Pgp-substrate interactions in realtime and in living murine cellular systems in which human Pgp have been expressed. Investigation of Pgp-substrate interactions is then extended to the structure-activity relationships (SAR) of potential Pgp substrates, on the basis of a recognition mechanism based on intramolecular hydrogen bond acceptor patterns. In the second part, living cultured murine cells were studied under the influence of cell penetrating peptides (CPPs). CPPs are a class of compounds that traverse biological membranes very efficiently, although the precise uptake mechanism is not yet known. Elucidation of the exact uptake mechanism is, however, of great interest, given the potential of this class of compounds for pharmacological and biotechnological purposes. A CPP derived from the HIV-1 peptide TAT was studied using fluorescence confocal microscopy and microphysiometry as principal techniques. In the third part of the thesis, the biological observation level was switched from living cells in culture to the entire organism using magnetic resonance imaging as a non-destructive method to observe biological effects of drugs. In pharmaceutical research the development of a commercial compound requires similar observation levels during the long evaluation process starting from high throughput screening of potential bioactive molecules and ending with the launch of one selected therapeutically successful product. The intermediate steps include basic in vitro studies, in vivo experimentation with cell culture, pre-clinical assays with intact animal models and phase I-III clinical trials with humans. For ethical and practical reasons, major attempts are made to replace invasive animal models by non-invasive techniques that can be applied in the same way to the clinical research providing thus a smooth transition between pre-clinical and clinical phases. This last part of the thesis adapts and optimizes such a recent imaging method to detect brain activation non-invasively in mice and to allocate the activated brain areas in mice without the need for surgery. In particular, the study focused on the activation of metabotrobic GABA(B) receptors in the mouse brain, upon stimulation with baclofen. The technical goal of this project was to adapt functional magnetic resonance imaging (fMRI) protocol at high field (7T) to the small size of the murine brain. The biological goal was to characterize regions in the brain, that show an enhanced activity under the influcence of baclofen - a know stimulator of GABAB receptors.