Problem: Penetration of a drug into the central nervous system (CNS) is vital for pharmacological efficacy if the target is located in the CNS, but represents an unwanted risk factor if the therapeutic target is peripherally located. CNS penetration is therefore required when it is known that the peripheral target exists in the CNS, or when and if any interactions with targets located or co-located in the CNS are likely to have undesirable consequences.

A commonly quoted index of brain penetration is the ratio of drug in brain tissue to that in circulation, i.e. [brain]/[plasma]. Obtaining such data in drug discovery can require a significant use of animals and is not resource effective when assessing the CNS penetration potential for multiple compounds. Furthermore, the concentrations of drug in brain and in plasma that are measured are most often the total concentration of drug in brain homogenate and total concentration of drug in plasma. But it is generally accepted that it is the free fraction of drug that is available for pharmacological activity and the difficult measurement is that of free drug in the brain.¹ Deriving this value from in vivo studies relies on technically challenging microdialysis or sampling of cerebrospinal fluid (CSF), techniques rarely available early in a drug discovery program.

Solution: Data from a number of in vitro models can be combined to assess the potential for molecules to penetrate the blood-brain barrier (BBB) and cause an effect in the CNS.

Under freely diffusible conditions it is expected that the free concentration in plasma and brain tissue will be the same, i.e.

[plasma]xfu_plasma = [brain]xfu_brain (where fu = fraction unbound)

Therefore from an efficacy perspective, there is no advantage in having a compound with a high [brain]/[plasma] ratio if the unbound drug concentrations in plasma are insufficient for efficacy. Conversely, if the unbound drug concentrations in plasma are sufficient for efficacy, this should also be true in brain tissue. The driving force for drug diffusion across the BBB is fu_plasmahence, the degree of drug penetration of the BBB is limited by high plasma protein binding (PPB). The fuplasma can be determined in vitro using equilibrium dialysis performed in 96-well format.

By combining the in vitro PPB data with the expected circulating plasma concentrations, an assessment of the free drug concentrations achievable in plasma can be made. This provides guidance on the likelihood of achieving efficacy (or side-effects) at a CNS target, assuming passive diffusion.

However, freely diffusible conditions may not exist if the drug has poor intrinsic cell permeability, or if active efflux takes place across the capillary endothelium. Cell lines such as Caco-2 or MDCK expressing the human MDR1 gene provide a measure of a compound’s intrinsic permeability and the potential impact of active transport. In particular, the efflux transporter, Pglycoprotein (MDR1), is highly expressed in brain microvessel epithelium, and confirmation of efflux by this protein can be obtained by the addition of inhibitors along with test compounds in the cell permeation assays.

If carrier-mediated uptake at the BBB occurs, fu_brain may be higher than fu_plasma. In the absence of such uptake, fu_brain is unlikely to be higher than fu_plasma and could be lower if active efflux of compound occurs. A guide to the amount of free drug in interstitial fluid can be obtained by measuring fu_brain in vitro using an equilibrium dialysis method with drug added to brain homogenate.

Therefore, assessing data from in vitro PPB studies, permeation assays and binding to brain homogenate provides an indication of the CNS penetration potential of test compounds.

For further information on the application of these techniques please contact BioFocus at info@glpg.com or visit www.biofocus.com.

1. De Lang, E. C.; Danhof, M. Considerations in the use of CSF pharmacokinetics to predict brain target concentrations in the clinical setting: implications of the barriers between blood and brain. Clin. Pharmacokinet 2002.41: 691-703