Abstract
Historically, the focus has been to use in vitro BBB models to optimize rate of drug delivery to the CNS, whereas total in vivo brain/plasma ratios have been used for optimizing extent. However, these two parameters do not necessarily show good correlations with receptor occupancy data or other pharmacological readouts. In line with the free drug hypothesis, the use of unbound brain concentrations (Cu,br) has been shown to provide the best correlations with pharmacological data. However, typically the determination of this parameter requires microdialysis, a technique not ideally suited for screening in early drug development. Alternative, and less resource-demanding methodologies to determine Cu,br employ either equilibrium dialysis of brain homogenates or incubations of brain slices in buffer to determine fraction unbound brain (fu,br), which is subsequently multiplied by the total brain concentration to yield Cu,br. To determine Cu,br/Cu,pl ratios this way, still requires both in vitro and in vivo experiments that are quite time consuming. The main objective of this study was to explore the possibility to directly generate Cu,br/Cu,pl ratios in a single in vitro model of the BBB, using a co-culture of brain capillary endothelial and glial cells in an attempt to mimick the in vivo situation, thereby greatly simplifying existing experimental procedures. Comparison to microdialysis brain concentration profiles demonstrates the possibility to estimate brain exposure over time in the BBB model. A stronger correlation was found between in vitro Cu,br/Cu,pl ratios and in vivo Cu,br/Cu,pl obtained using fu,br from brain slice than with fu,br from brain homogenate for a set of 30 drugs. Overall, Cu,br/Cu,pl ratios were successfully predicted in vitro for 88% of the 92 studied compounds. This result supports the possibility to use this methodology for identifying compounds with a desirable in vivo response in the CNS early on in the drug discovery process.
Highlights
The market for neuropharmaceuticals is regarded as one of the potentially largest sectors of the global pharmaceutical market owing to the increase in average life expectancy and that many neurological disorders have been largely refractory to pharmacotherapy.The value of many promising CNS drug candidates is diminished by at the level of the cerebral capillaries of the blood-brain barrier (BBB), which possess both structural and enzymatic components
Total brain/plasma ratios was for a long time the dominating parameter used for optimizing CNS exposure despite the fact that this parameter often show a poor correlation with receptor occupancy data or other pharmacodynamic readouts [3]
The possibility to generate in vitro Cu,br/Cu,pl ratios at 1 hour and to predict Cu,br/Cu,pl ratio under steady-state conditions in one single experiment in a BBB model represent a great simplification over existing experimental procedures
Summary
The market for neuropharmaceuticals is regarded as one of the potentially largest sectors of the global pharmaceutical market owing to the increase in average life expectancy and that many neurological disorders have been largely refractory to pharmacotherapy.The value of many promising CNS drug candidates is diminished by at the level of the cerebral capillaries of the blood-brain barrier (BBB), which possess both structural and enzymatic components. To have an in vitro model that can be used for predictions of brain uptake and investigations of therapeutic interventions at the level of the cerebral capillaries is highly advantageous This provides powerful means to assess the risk for taking compounds further in the pharmaceutical development process, and generates important information to allow for rational drug design [1]. These are fairly straightforward techniques for determining Cu,br, both in vitro and in vivo experiments are required, which are quite time-consuming Additional caveats with these techniques would be if the homogenate and slice methods generate significantly different results in terms of fu,br, which in turn lead to different estimations of free brain levels. Previous reports suggest for instance that variations in experimental design (homogenization, incubation times, slice thickness etc.) may influence the outcome of the experiments [8]
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