Figure 1.
Schematic diagram of the experimental protocol.
After 12 days of coculture in cell culture medium, both compartments of the in vitro BBB model consisting of a coculture of brain capillary endothlial cells and rat primary glial cells were filled with Hepes-buffered Ringer’s solution and the compounds dissolved at 2 µM were added in the luminal compartment (i.e donor). The presence of glial cells in the abluminal compartment was used to reproduce the non specific binding to brain tisssue within the brain prenchyma in vivo. After 1 hour, aliquots were taken from both compartments. In vitro Cu,br/Cu,pl at 1 h were calculated by dividing the concentration in the abluminal compartment (∼Cu,br) by the concentration in the luminal compartment (∼Cu,pl) at 1 h. The experimental data at 1 h were computed using the blue-norna steady-state calculator to generate the in vitro steady-state Cu,br/Cu,pl.
Table 1.
Administration parameters used for brain microdialysis experiments.
Figure 2.
Free brain and plasma concentrations - time profile of three commercial compounds.
Sulpiride (A), Risperidone (B), Clozapine (C) and 3 proprietary compounds (D, E and F) determined by brain microdialysis and compared with free brain concentrations obtained by multiplying the free plasma concentration from the microdialysis experiment with Cu,b/Cu,p ratios obtained in vitro using the in vitro BBB model. Free plasma concentration at earlier time point than 1 hour were used together with in vitro Cu,br/Cu,pl ratio at 1 h and microdialysis plasma concentration data at 2 hours and beyond were multiplied by the in vitro steady state ratio to simulate the microdialysis profile. Values for drugs are defined in Table 2 and values for proprietary compounds in Table 3.
Table 2.
Brain unbound fractions obtained using either the brain slice or the brain homogenate method and in vitro and in vivo unbound brain/plasma ratios of 30 drugs.
Table 3.
In vivo and in vitro Cu,br/Cu,pl of 62 proprietary compounds.
Figure 3.
Relationship between in vivo Cu,br/Cu,pl obtained using fu,b from either brain slice or brain homogenate (A); Relationship between in vitro predicted steady-state Cu,br/Cu,pl and in vivo Cu,br/Cu,pl obtained using fu,b from either brain slice (B) or brain homogenate (C).
The solid line represents perfect agreement. The dashed lines represent a 2-fold over- or underestimation compared with in vivo Cu,br/Cu,pl.
Figure 4.
Relationship between in vitro predicted steady-state Cu,br/Cu,pl and in vivo Cu,br/Cu,pl obtained using fu,b from brain slice for 30 drugs and 62 proprietary compounds.
The dashed lines represent a 2-fold over- or underestimation compared with in vivo Cu,br/Cu,pl. Individual values for drugs as well as proprietary compounds are defined in Table 2 and 3.
Figure 5.
Comparisons between predicted free brain exposures (from Cu,br/Cu,pl in vitro) and in vivo exposures in relation to estimated IC50 in brain for AZ13032000 = 55 nM based on in vitro potency data.