Validation of in vitro Blood-Brain Barrier Models

Research project P 14582 Validation of in vitro Blood-Brain Barrier Models Christian R. NOE 09.10.2000 Glutamate is a basic neutrotransmitter in the brain, acting on a class of receptors. Among these, the NMDA- receptor has achieved specific attention not only due to the fact that the study of its complex structure and function has remained a major target of neuroscience, but also to its central role in physiological processes such as learning and memory. Not surprisingly, the NMDA receptor has been associated a series of deseases of the brain, such as epilepsy, Parkinson-desease, dementia, stroke and others). It is an important target for drug research. It is a specific property of the NMDA-receptor that in addition to the agonist glutamate and several modulators (e.g. spermine, magnesium) there is a co-agonist, glycine, required for its proper function. The glycine-binding site has attracted interest of several research groups. High affinity ligands at this site may be used as tools in the study of the structure of this part of the NMDA-receptor, but may also be promising candidates in drug development. During the last few years high affinity ligands have been synthesized, one of these `compounds even has reached clinical phase 111. Nevertheless, it is still not very well understood, why there is no good correlation between receptor affinity of such compounds and their pharmacological activity in the brain. It is evident that the blood brain barrier is involved leading to an impaired bioavailability, but simple models based on lipophilicity do not explain the effects sufficiently. The transfer of compounds from the blood into the brain interstitial fluid is regulated by the function of brain capillary endothelial cells. The aim of the project is to establish a several blood brain barrier models based on determination of physicochemical parameters, culturing of endothelial cells and calculation of suitable descriptors. These models will be thoroughly validated based on !published in vivo data. Subsequent statistical analysis based on principal component and partial least squares analysis should lead to a general model capable of proper predicting blood- brain barrier permeation. This model will guide our efforts on the development and optimization of new cns-active drugs, in particular putting forward our investigations on glycine antagonists acting on the NNMA receptor. Additionally, using closely related compound libraries, criteria for influx and efflux of compounds will be investigated on a molecular basis.

The blood-brain barrier (BBB) separates the brain and central nervous system (CNS) from the blood stream. Therefore, in CNS drug development it is of vital interest that the compounds are able to cross the BBB. Conversely, compounds designed for non-CNS targets should not cross the BBB to avoid unwanted side effects. Although the study of the blood-brain barrier is a relevant topic in physiological and pathophysiological research with several BBB-models reported, there is at present no standard model available, which is validated with respect to serving as a tool in the hit to lead process. This project thus focused on the development and, most important, on the validation of different in vitro blood-brain barrier models for lead compound selection in an early stage of the CNS-drug discovery process. Within this project, the following main results have been achieved: A protocol for Transwell-based in vitro systems was established which improves reproducibility of permeability values and form the basis for generation of validated data sets. These are heavily needed as basis for generation of predictive in silico models. An APTS-Labeled Dextran Ladder was developed which allows to characterise cell layer tightness in detail and to correlate molecule-based tightness markers to TEER-values. Up to now tightness of cell layers was measured either via the electrical resistance or via FITC-dextran or sucrose as marker. The APTS-labeled dextran ladder developed within this project allows for the first time a direct correlation between electrical resistance values and leakiness on a molecular basis. A dynamic in vitro model of the BBB was developed and optimized to be suitable in the lead optimization process. Establishing a dynamic system mimics much closer the in vivo situation with a continuous blood stream and a concentration gradient.