Conclusions for BBB InVitro Models

Recent advances in combinatorial chemistry and HTS for pharmacological activity have greatly expanded the number of drug candidates for the therapy of neurological diseases. Rapid screening for BBB penetration early in the drug discovery phase can streamline the quest for promising lead compounds, and provide guidance for the rational design and synthesis of novel compounds targeting the CNS. The tight barrier of the specialized endothelial cells forming/ lining the BBB will prevent most drugs from entering the brain. However, those compounds that do penetrate the BBB enter the CNS by transcellular passive diffusion. Over the past three decades, several in-vitro BBB biological models have been developed using transwell tissue culture inserts, which can accommodate co-cultures of brain microcapillary endothelial and glial cells, primary or cloned, from different origins. Whilst measurements of the TEER are useful as a concept, their value in these simple in-vitro BBB models in terms of predicting the in-vivo BBB permeability of specific drugs is very limited. Hence, the reliability of these first generations of in-vitro BBB models is questionable. At present, two in-vitro

BBB biological models of bovine and porcine origin, are available, which show highly restrictive paracellular permeability properties and are more closely related to the physiological parameters measured in the BBB in situ. However, at the time of writing, no model - either static or dynamic - can reliably and reproducibly provide adequate TEER- and P-values of CNS drugs. In our view, despite all past and current efforts, no optimal in-vitro BBB pharmaceutical model exists to date, which not only reflects our lack of detailed knowledge of the BBB but also indicates the need to use novel tissue engineering approaches to generate such a model in vitro.

These novel approaches will comprise: (1) intelligent scaffolds mimicking the structure/function and regional heterogeneity of the brain ECM; (2) novel HTS devices, which will incorporate flow and hydrostatic pressure as well as online TEER measurements; and (3) on-line concomitant sampling of the media, on both sides of the insert, to measure drug permeability.

There is an urgent need for high-quality, human cells to generate the in-vitro BBB model. This need can most likely be met by utilizing human brain endothelial, glial and neuronal stem/precursor cells. In our view, the optimal future model will comprise a hemodynamic system that will integrate synthetic CNS-compatible biomaterials as insert coating, together with human stem cell-derived glia and neurons and brain microvessel ECs, which can be induced to stably express the BBB phenotype (including abundant tight junctions). This future model will have to be validated for a large number of known CNS drugs, and to be compared with in-vivo measurements to establish the obligatory pharmaceutical in-vitro-in-vivo correlation (IVIVC) in order to be adopted by pharmaceutical companies and the Food and Drugs Administration (FDA). During the next decade of tissue engineering, we anticipate the emergence of fascinating, new, high-fidelity in-vitro BBB models, together with in-silico artificial membrane permeation assays (PAMPA-BBB [221]), and the generation of vast databases of CNS drugs. These advances will finally open the current bottleneck in the development of novel, more effective CNS drugs for the benefit of patients.

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