Combining PET biodistribution and equilibrium dialysis assays to assess the free brain concentration and BBB transport of CNS drugs

Abstract

The passage of drugs in and out of the brain is controlled by the blood-brain barrier (BBB), typically, using either passive diffusion across a concentration gradient or active transport via a protein carrier. In-vitro and preclinical measurements of BBB penetration do not always accurately predict the in-vivo situation in humans. Thus, the ability to assay the concentration of novel drug candidates in the human brain in vivo provides valuable information for de-risking of candidate molecules early in drug development. Here, positron emission tomography (PET) measurements are combined with in-vitro equilibrium dialysis assays to enable assessment of transport and estimation of the free brain concentration in vivo. The PET and equilibrium dialysis data were obtained for 36 compounds in the pig. Predicted P-glycoprotein (P-gp) status of the compounds was consistent with the PET/equilibrium dialysis results. In particular, Loperamide, a well-known P-gp substrate, exhibited a significant concentration gradient consistent with active efflux and after inhibition of the P-gp process the gradient was removed. The ability to measure the free brain concentration and assess transport of novel compounds in the human brain with combined PET and equilibrium dialysis assays can be a useful tool in central nervous system (CNS) drug development.

Figure 1

Magnetic resonance imaging (MRI) (A) and positron emission tomography (PET) (B, C) images of the Yorkshire-Landrace Pig brain in transaxial, sagittal, and coronal sections (MRI and PET data are obtained from different animals). (A) Structural T1-weighted MRI scans for anatomical reference. (B, C) [¹¹C]Loperamide PET scans (integral images 0 to 90 minutes) displayed in standardized uptake units (%ID/L); panel B is at baseline and indicates very little uptake for the known P-glycoprotein (P-gp) substrate, whereas panel C is after administration of cyclosporin-A (30 mg) which inhibits the P-gp efflux and allows for brain penetration of [¹¹C]Loperamide. In both (B) and (C), the pituitary, a structure which exists outside the blood–brain barrier (BBB), is visible and shows uptake of [¹¹C]Loperamide.

Figure 2

Positron emission tomography (PET) arterial plasma parent input functions for the (n=36) radiolabelled compounds. For [¹¹C]Loperamide just the baseline input function is displayed.

Figure 3

Positron emission tomography (PET) cerebellar time activity curves (TACs) for the (n=36) radiolabelled compounds (○) and model fits from the parsimonious compartmental analysis (−). For [¹¹C]Loperamide two scans are shown, one at baseline (○) and the other after administration of 30 mg cyclosporin-A ( × ) which inhibits P-glycoprotein (P-gp).

Figure 4

Comparison of in-vitro (equilibrium dialysis free fraction ratio—f_P/f_ND) and in-vivo (positron emission tomography (PET) nondisplaceable distribution volume—V_ND) measures. The symbols used indicate whether the in-silico modelling predicted that they were more likely to be P-glycoprotein (P-gp) substrates (•) or whether they were more likely to undergo passive diffusion (○). (A) Plot of equilibrium dialysis versus PET. (B) Bland-Altman plot of data in panel A using data from the compounds predicted in-silico to undergo passive diffusion (○) to define the confidence intervals (μ denotes the mean). In both plots, the data connected by the dashed line identify the results from the two [¹¹C]Loperamide scans (baseline (•) and the other after administration of 30 mg cyclosporin-A (○) which inhibits P-gp).