Assessment of Blood–Brain Barrier Permeability Using the In Situ Mouse Brain
J. Cory Kalvass,
and Gary M. Pollack
Received October 17, 2008; accepted March 16, 2009; published online April 22, 2009
Purpose. To assess the blood–brain barrier (BBB) permeability of 12 clinically-used drugs in mdr1a(+/+)
and mdr1a(−/−) mice, and investigate the influence of lipophilicity, nonspecific brain tissue binding, and
P-gp-mediated efflux on the rate of brain uptake.
Methods. The BBB partition coefficient (PS) was determined using the in situ mouse brain perfusion
technique. The net brain uptake for 12 compounds, and the time course of brain uptake for selected
compounds ranging in BBB equilibration kinetics from rapidly-equilibrating (e.g., alfentanil, sufentanil)
to slowly-equilibrating (fexofenadine), was determined and compared.
Results. There was a sigmoidal relationship in mdr1a(−/−) mice between the log-PS and clogD
range of 0–5. The brain uptake clearance was a function of both permeability and blood flow rate. The
brain unbound fraction was inversely proportional to lipophilicity. Alfentanil achieved brain equilibrium
approximately 4,000-fold faster than fexofenadine, based on the magnitude of PS×fu,brain.
Conclusions. In situ brain perfusion is a useful technique to determine BBB permeability. Lipophilicity,
ionization state, molecular weight and polar surface area are all important determinants for brain
penetration. The time to blood-to-brain equilibrium varies widely for different compounds, and is
determined by a multiplicity of pharmacokinetic factors.
KEY WORDS: blood–brain barrier; permeability; protein binding; time to equilibrium.
The blood–brain barrier (BBB) is a continuous layer of
brain capillary endothelial cells, connected by highly-devel-
oped tight junctions, that expresses numerous efflux trans-
porters and metabolizing enzymes. The BBB represents a
formidable barrier to the access of therapeutic targets for
agents intended to have central nervous system (CNS)
activity (1–4). Physicochemical properties (e.g., molecular
weight, pKa, lipophilicity, polar surface area, and the number
of hydrogen bonds) as well as biological factors (e.g., plasma
and brain tissue binding, affinity to transporter systems at
the BBB) are known determinants of substrate flux across
the luminal and abluminal membranes of brain capillary
endothelial cells (5–8). Whereas the role of other efflux
transporters at the BBB remains unclear, P-glycoprotein
(P-gp)-mediated efflux of many therapeutic agents has been
widely accepted (4,9). P-gp is a 170-kDa membrane protein
encoded by multidrug resistance gene MDR1 in human and
mdr1a and mdr1b in rodents. P-gp-mediated reduction in
brain exposure has been linked to decreased CNS pharma-
cologic or toxic effects (9,10).
The logarithm of the brain-to-blood (or brain-to-plasma)
concentration ratio (logBB) often is used to characterize and
predict BBB permeation. However, it is recognized that the
unbound brain concentration is the relevant driving force for
most pharmacologic events in the CNS. Because logBB is a
hybrid parameter that is determined by permeability, plasma
and brain tissue binding, and active transport mechanisms, it
may provide misleading information regarding potential in
vivo pharmacologic response, thereby excluding potentially
useful drug candidates from further consideration (11,12).
The log of the permeability-surface area coefficient (logPS),
in contrast, measures the ability of a drug to cross the BBB
and move into brain tissue over time, and is a more relevant
metric of the rate of BBB penetration (11–13).
PS can be measured using an in vivo or in situ short-
duration vascular perfusion method, e.g., brain uptake index,
indicator dilution, or in situ brain perfusion (14). In situ brain
perfusion was developed to provide a simpler and more
accurate method to determine BBB permeability compared
2009 Springer Science + Business Media, LLC
Pharmaceutical Research, Vol. 26, No. 7, July 2009 (
Division of Pharmacotherapy and Experimental Therapeutics,
University of North Carolina at Chapel Hill, Chapel Hill, NC
Drug Disposition, Eli Lilly and Company, Indianapolis, IN 46285,
School of Pharmacy, University of North Carolina at Chapel Hill,
Chapel Hill, NC 27599-7360, USA.
To whom correspondence should be addressed. (e-mail: gary_
ABBREVIATIONS: BBB, blood–brain barrier; clogD
octanol–water partition coefficient at pH 7.4; clogP, calculated
octanol–water partition coefficient in unionized state; Cl
clearance; CNS, central nervous system; f
, brain unbound
fraction; P-gp, P-glycoprotein; PS, permeability-surface area
product; RSD, relative standard deviation.