J Pharmacokinet Pharmacodyn (2013) 40:301–314
DOI 10.1007/s10928-013-9303-7
REVIEW PAPER
In vitro, in vivo and in silico models of drug distribution
into the brain
Scott G. Summerfield • Kelly C. Dong
Received: 11 November 2012 / Accepted: 31 January 2013 / Published online: 13 February 2013
Ó Springer Science+Business Media New York 2013
Abstract Achieving sufficient brain penetration to elicit
efficacy in humans is one of the most challenging tasks for
scientists in CNS Drug Discovery. Substantial progress has
been made in the past decade in understanding the factors
influencing the rate and extent of brain distribution via a
variety of in vivo, in vitro and in silico methodologies, and
hence, predict their likelihood of success in man. This
purpose of this review is to summarize the current
approaches with a special focus on parameters related to
free drug concentrations in brain which are the most
pharmacologically relevant for the majority of CNS disease
targets. Due to the dynamic and complex nature of this
targeted organ, it is inevitable that these approaches have
not been able to provide a fully comprehensive assessment
of brain distribution and are expected to evolve further in
the years to come.
Keywords Brain  Distribution  In vivo  In vitro 
In situ  In silico  PET
Introduction
Passage across the blood-brain barrier (BBB) and distri-
bution into brain parenchyma are key steps for drugs acting
on receptors within the central nervous system (CNS). The
BBB comprises a tight junctioned cellular structure that
S. G. Summerfield (&)
GlaxoSmithKine R&D, Sir David Jack Research Centre,
Ware SG12 0DP, Hertfordshire, United Kingdom
e-mail: scott.g.summerfield@gsk.com
K. C. Dong
GlaxoSmithKine R&D China, Zhangjiang Hi-tech Park,
Shanghai 201203, China
virtually eliminates paracellular diffusion in the healthy
brain [1]. Expressed within the BBB are also a range of
transport systems whose role is to either supply CNS tissue
with the necessary nutrients for maintenance of brain
homeostasis or to act as gatekeepers and expel potentially
undesirable xenobiotics [2]. Therefore for drugs to traverse
the BBB effectively they must pass either by means of
transcellular diffusion or be recognised by an uptake
transporter.
Designing in the necessary properties to meet adequate
potency, pharmacokinetics and also facilitate distribution
into the brain is an exceptionally challenging task. Attrition
of potential CNS agents during Drug Development is
among the highest of all common therapeutic areas [3]. As
noted recently by Morgan et al. [4], the chances of making
the right decision to progress or terminate a clinical drug
candidate are enhanced when one can demonstrate the
presence of drug in the biophase, have the verified target
engagement and that this leads to the expected pharma-
cology. Drug metabolism and pharmacokinetics (DMPK)
has a role to play in all three of these ‘pillars’ although the
aim of this article is to focus on the first, namely having
techniques to show that the drug is distributed into the
brain. Indeed this is the necessary precursor to the other
pillars being met.
Figure 1 shows a schematic representation of the inter-
relationship between the blood and brain compartments
following administration of a potential CNS agent. Drug in
blood is in equilibration between the unbound fraction and
the fraction bound to plasma proteins and blood cell con-
stituents. The unbound portion is available to undergo
distribution into tissues or undergo elimination from the
body, e.g. via hepatic and renal clearance. At the level of
the brain, if the drug is sufficiently lipophilic then the
unbound portion will be able to dissolve into the cellular
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environment of the BBB and pass through to the brain
extracellular fluid (ECF) by means of passive transcellular
diffusion. Unbound drug in the blood may also interact
with transporters at the BBB surface to facilitate their
passage [5, 6]. Once distributed into brain ECF then
unbound drug may distribute further into the brain paren-
chyma, either binding to the target, other receptors or non-
specifically to surrounding lipophilic brain tissue. A variety
of measures of brain distribution can be taken either in
terms of concentration (unbound or total), as a partition
ratio between brain and blood or plasma (unbound or total)
or as a clearance across the BBB (unbound). There are also
in vitro or in silico equivalents available to Drug Discovery
scientists for all of these measurements. The unbound
fraction in blood (or plasma) may be determined by means
of equilibrium dialysis, ultrafiltration or microdialysis [7].
Similarly the unbound brain fraction may be estimated via
equilibrium dialysis of brain homogenate [8, 9], brain tis-
sue slices [10, 11], cerebrospinal fluid (CSF) [12] or brain
microdialysis [13, 14]. The rate of drug passage across the
BBB may be measured directly using in situ brain perfu-
sion [5] or in vitro using cell-based methods [15] or arti-
ficial membranes [16, 17]. Concentrations within the brain
can be also measured either as a total value (e.g. the sum of
the unbound and bound fractions) by homogenising excised
brain tissue, or alternatively, unbound by means of brain
microdialysis [13, 14]. Few techniques have the capability
to capture most or all of these parameters within a single
experiment. The closest are in vivo microdialysis and
temporal imaging techniques like positron emission
tomography (PET) which provide significant benefits in
this regard [18, 19].
Figure 1 also demonstrates the importance of the
unbound concentration in determining the passage and
distribution of drugs to and within the brain, which aligns
well to the fact that is also the fraction available to bathe
receptors in the appropriate cellular compartment. Many of
Fig. 1 Schematic of drug distribution into the brain from the blood
compartment. Ctotal and Cu denote the total and unbound concentra-
tions in blood. Concentrations in the brain are shown in extracellular
fluid (ECF) and cerebrospinal fluid (CSF). Dotted lines show the
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J Pharmacokinet Pharmacodyn (2013) 40:301–314
the more recent in vitro and in silico developments have
been centred on unbound brain parameters, which have
helped shift the focus in Drug Discovery away from
measurements based on total concentrations that are less
relevant with respect to distribution mechanisms and
pharmacological activity. This review aims to cover the
key elements of total and unbound measurements and
where they make the greatest impact on the discovery
effort for new CNS medicines.
In vivo methodologies
Unbound concentration measurements
Brain microdialysis allows for the temporal monitoring of
brain ECF concentrations in concert with the blood or
plasma pharmacokinetics [13, 14] and can also be used to
track changes in neuro biomarkers [20]. Taking measure-
ments from multiple compartments enables all of the
ensuing information to be woven together through physi-
ologically based pharmacokinetic (PBPK) modelling at the
level of the brain [21, 22]. However, this technique requires
the most surgical skill of all the platforms of in vivo
methods available. A dialysis probe is inserted stereotaxi-
cally into the brain after exposing it through a hole in the
cranium. Dialysis fluid is pumped slowly through the probe
whereupon it meets the brain ECF across a semi-permeable
membrane at the probe tip. The composition of this dialysis
fluid must match that of ECF in order to maintain the
osmotic balance and homeostasis within the brain.
Unbound drug diffuses across this membrane and flows
back along the probe tubing where it is collected continu-
ously into sampling vials. Tracking the brain kinetics of
drugs and their metabolites has been employed to under-
stand efficacy discrepancies for agents such as clozapine
and morphine [23, 24]. Following on the work with brain
blood-brain barrier (BBB) and blood-CSF barrier (BCSFB). Drug
distribution is depicted by the total concentration ratio Kp and
unbound ratio Kp,uu and the rate of passage across the BBB as Kin
J Pharmacokinet Pharmacodyn (2013) 40:301–314
microdialysis, Gupta et al. [25] introduced the concept of
Kp,uu or the unbound brain-to-unbound plasma concentra-
tion ratio. In essence, Kp,uu measures the unbound con-
centration gradient across the BBB, which would equal
unity for drugs undergoing passive transfer. Similarly, a
Kp,uu value less than unity would suggest passage across
the BBB is restricted by either inherently low permeability
(e.g. atenolol arising from its physicochemical properties)
or by active efflux through a transporter, such as P-glyco-
protein (P-gp). A Kp,uu value greater than one would
indicate that uptake into the brain is facilitated in some
way, perhaps via active influx.
In addition to the severe surgery associated with inser-
tion of a microdialysis probe, the resulting brain ECF is
highly diluted within the dialysate flow. This dilution effect
and the absence of protein or lipid within the dialysate
often leads to adsorptive losses of the analyte over the
internal surfaces of the probe and tubing, thus necessitating
calibration methods to compensate for these losses [26]
together with highly sensitive bioanalytical measurements.
Taken together with the low throughput and high probe
costs, these experimental challenges restrict the utility of
brain microdialysis to more bespoke studies rather than
being deployed as a general screening tool. However, it is
clear that a great deal of key information on brain dispo-
sition is attainable if the effort is put into running these
experiments.
Sampling CSF has been proposed as a means of taking a
surrogate unbound concentration measurement for brain
ECF [12]. CSF is produced continuously by the choroid
plexus and cushions the cortex from severe head move-
ments and in rats the CSF volume is ca. 300 uL and flows
at ca. 2 uL/min [27, 28]. For compounds where BBB dif-
fusion is both rapid and passive then CSF concentrations
agree well with brain ECF concentrations [12]. However
there are instances where CSF and ECF brain concentra-
tions do not correlate well such as when the drug is subject
to a slow rate of uptake across the BBB, active transport or
local metabolism [28–30]. Generally, CSF concentrations
appear to be a better surrogate of unbound brain concen-
trations than using unbound plasma concentrations [12].
Indirect determination of unbound brain concentrations
may be achieved by first measuring the total brain tissue
concentration and correcting this with the in vitro unbound
brain fraction [8]. Despite the need for two measurements,
this approach is far more amenable to screening and tri-
aging the greater numbers of compounds within the Drug
Discovery setting. Excision of brain tissue and the sub-
sequent homogenisation can be mastered by a wider range
of scientists and technicians than the surgical expertise
required for microdialysis. In addition the unbound fraction
methods are amenable to moderate automated throughput.
However, microdialysis can provide temporal information
303
via serial sampling in the same animal whereas it is not
possible when total tissue concentrations are measured and
then corrected to unbound values because brain excision is
a terminal procedure. This means numerous additional
animals are required to create a time course but often this
compromise is paid for the simplicity of the approach.
The accuracy of the total concentration measurement
relies on having complete tissue extraction and no inter-
ferences during the bioanalysis but there is actually little
information available to support how well drugs are
extracted from brain tissue. For bioanalytical techniques
such as liquid chromatography–tandem mass spectrometry
(LC–MS/MS) calibration standards and quality control
samples are typically prepared by spiking analyte into brain
homogenate whereas from the in vivo experiment, the drug
resides within the parenchyma and requires efficient
extraction in order to reflect the associated standards.
Supporting information for the efficient drug extraction
could come from comparing fubrain values derived from
spiking drug into brain homogenate versus drug dosed
in vivo and then brain tissue subsequently homogenised
and being subjected to dialysis (Table 1). The close con-
cordance between the in vitro and ex vivo measurements
suggests that the combination of tissue homogenisation and
solvent extraction is effective at releasing the drug.
Total concentrations measurements
The ease of measuring total brain concentrations has led to
brain distribution often being quoted in terms of the par-
tition ratio (Kp), which is defined as the total concentration
in brain divided by the total concentration in blood (or
plasma). It is now generally accepted that Kp values rep-
resent a rather blunt instrument for interpreting whether a
compound is ‘good’ or ‘poor’ in terms of brain penetration
or indeed efficacy [27] but can be informative with in vivo
models to characterise active transport at the level of the
BBB [31, 32]. In these models the drug is administered to
naive rats or mice and then again to animals where the
transporter of interest (generally P-gp) has been inactivated
either chemically [33, 34] or genetically [31, 32]. Kalvass
and Maurer [8] showed that the unbound brain fractions of
several P-gp substrates were identical in wild-type and
P-gp knockout mice [31] and hence the subsequent changes
in Kp are the result of altering BBB transport (e.g. a rate
effect) rather than altering binding (e.g. an extent effect).
This leads to the establishment of an unbound concentra-
tion gradient reflected in Kp,uu. Murine gene knockouts of
other putative BBB transporters are also available for the
study of brain distribution in selective cases [35–41] and
the changes observed are far lower (generally less than
two-fold) than those noted for P-gp substrates in the mdr
1a/b(-/-) model. Many P-gp substrates are lipophilic
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Table 1 Unbound brain fractions measurements taken from in vitro studies (control brain homogenate spiked with analyte) and in vivo studies
(iv administration followed by brain excision and homogenisation)
Unbound brain (% free)
Cpd 1
Rat Aa 5.8 ± 0.6
Rat Ba 4.8 ± 0.4
Rat Ca 4.2 ± 0.4
Spiked brain (fresh tissue)b 4.8 ± 1.0
Spiked brain (frozen tissue)b 4.6 ± 0.7
NT not tested
a
Drug administered by intravenous infusion with brain excision, homogenisation and extraction performed according to Ref. [9]
b
Drug spiked into control brain tissue at a concentration of 1 ug/mL [9]
bases and so would be expected to undergo extensive tissue
distribution when freely permeable across the BBB of the
knockout strain. In contrast, the physicochemical proper-
ties of substrates for several of these BBB transporters may
limit their inherent brain distribution, which may help to
explain the small increases observed. In higher species
information is far rarer on BBB transporter differences
across breeds and hence chemical knockout is generally
employed as an adjunct to imaging techniques such as PET
[42, 43]. One example of P-gp deficiency has been iden-
tified in Collie dogs [44] although it is unclear whether any
advantage is conferred over the chemical intervention.
Aside from these instances, terminal brain penetration
measurements in higher species are performed less fre-
quently because these species are often reused in pharma-
cokinetic studies and are employed less commonly for
pharmacology models of CNS conditions. Before much of
the work cited here was published, our own company, like
many others, was focusing on Kp measurements. Cross
species information on Kp was thought to be useful since
the entire clinical premise was based on the brain pene-
tration in rodents. So a series of brain penetration experi-
ments were performed in pigs because of their use in PET
biodistribution studies and pharmacokinetic models for
scaling. Following 1 h iv infusions, the brains were excised
and several brain regions were dissected out in order to
measure the total concentrations (Table 2). Overall the
regional Kp values were very close and no substantial
differences in brain regions were noted. Essentially the data
showed that in most instances the Kp in rats and pigs were
different but it was not clear why. Compound A had also
been identified as a P-gp substrate and Kp was measured in
several other species (Table 3). Although the brain pene-
tration was marginally higher in rats, generally the data
across species added nothing more. The advent of brain
tissue binding screens and an explicit description for the
roles of plasma and tissue binding by Kalvass and Maurer
[8] paved the way for a more directed approach toward
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Cpd 2 Cpd 3 Acetaminophen
2.1 ± 0.3 0.080 ± 0.016 68.5 ± 18.5
2.0 ± 0.3 0.080 ± 0.044 75.4 ± 26.8
2.1 ± 0.3 0.089 ± 0.054 85.1 ± 21.1
2.0 ± 0.1 0.075 ± 0.008 72.0 ± 5.0
2.1 ± 0.1 NT NT
understanding brain distribution based on the role of
unbound drug. Species differences in Kp could be shown to
arise from changes to blood/plasma binding [9] and today
this might also be seen with the measurements of Kp,uu if
active transport is implicated. Subsequently, a collabora-
tive effort was initiated with a PET imaging centre and a
series of pig PET biodistribution studies were performed
and complemented with in vitro plasma and brain tissue
binding data. Generally in vitro estimates of brain distri-
bution (e.g. fuplasma/fubrain) were comparable to in vivo
estimates from the tracer experiments indicating passive
diffusion across the BBB for many of the compounds
tested [45]. Loperamide, a known substrate for P-gp, was
characterised by a low in vivo Kp value in the pig but
showed good agreement with the in vitro Kp measurement
when co-administered with cyclosporine due to inhibition
of the P-gp effect.
Total brain concentration measurements do not neces-
sarily just provide information on the extent of brain pen-
etration; they can also be used to derive information on the
rate of drug passage across the BBB when used in con-
junction with in situ brain perfusion studies [5, 46, 47].
Generally performed in rodents, in situ brain perfusion
involves the administration of drug via rapid perfusion into
the carotid artery (\1 min at a flow of 5–10 mL/min) to
surgically prepared animals. The brain is excised and the
total brain tissue concentrations are determined by means
of radioactivity [5] or LC–MS/MS [47]. Under this short
perfusion paradigm drug transfer across the BBB is con-
sidered to be the dominant process while back-flux of drug
to the vascular compartment is negligible under these
conditions [5]. In order to benchmark compounds across
experiments several control compounds are generally
included and this has provided an excellent and reliable
data set across studies reported in the literature. A non
brain penetrant agent is added to the perfusion solution in
order to ascertain and correct for the vascular volume (e.g.
14
C sucrose or atenolol). Antipyrine is often used as a
J Pharmacokinet Pharmacodyn (2013) 40:301–314 305

Table 2 Brain distribution for 14 compounds measured in terms of Kp in rodents (whole brains) and pigs (regional values)
Cpd Rat Pig brain Kbp
Kap
Mean Dorsal Cingulate and Hippocampus Caudate Nucleus Hypothalamus Cerebellum Rest Cervical
lateral infralimbic putamen accumbens of spinal
cortex cortex brain cord

A 0.28 0.05 0.04 0.04 0.05 0.05 0.05 0.05 0.06 0.05 0.04
B 1.53 4.16 4.56 4.53 4.06 4.14 4.29 4.17 3.63 3.93 4.06
C 0.50 1.07 0.94 1.12 1.24 0.86 1.22 1.08 1.09 1.01 1.18
D 0.20 0.51 0.57 0.51 0.50 0.50 0.47 0.50 0.54 0.50 0.27
E 5.00 11.01 13.52 12.23 11.45 9.70 10.77 10.01 9.95 10.46 6.54
F 3.25 4.50 4.92 4.39 4.29 4.24 4.47 4.56 4.52 4.58 2.01
G 3.16 6.80 6.72 6.66 6.96 6.32 6.70 6.94 6.77 7.30 4.43
H 0.14 2.04 2.36 1.99 1.69 2.35 2.10 1.69 2.41 1.74 2.08
I 0.66 1.96 2.04 1.81 1.94 1.94 1.91 2.03 1.94 2.07 2.17
J 0.80 1.37 1.34 1.35 1.38 1.44 1.42 1.30 1.48 1.25 1.45
K 1.53 5.57 6.27 5.65 5.80 5.56 5.62 5.68 4.51 5.51 3.74
L 3.45 14.49 16.13 15.83 14.00 14.13 14.47 14.02 12.80 14.54 8.96
M 0.80 0.91 0.68 1.15 1.08 0.70 1.02 0.64 1.00 1.04 0.87
N 0.50 0.29 0.34 0.29 0.29 0.21 0.25 0.26 0.34 0.31 0.21
a
Steady-state iv infusion. A dose of 1 mg/kg was administered in all cases
b
1 h iv infusion. A dose of 1 mg/kg was administered in all cases

Table 3 Brain distribution for three compounds measured in terms of When comparing Kin or PS values for different
Kp across a range of preclinical species (no significant regional
compounds it is important to remember that these
differences)
parameters focus on the rate of uptake and provide no
Cpd Species Kap information on the extent of distribution. This is
Rat Minipig Landrace pig Monkey Dog Marmoset exemplified in Fig. 2a, b which show Kin [47] plotted in
the order of increasing Kp (steady-state) and Kp,uu values.
A 0.28 0.05 0.06 0.05 0.11 0.05
Correlations in both instances are poor because Kp and
B 1.53 4.16 1.73 1.07 3.05 3.80
Kp,uu are partition ratios and thus provide no detail on the
C 0.5 1.07 0.68 0.40 1.02 0.64
rate of drug uptake into the brain. Extent and rate
a
1 h iv infusions for all species except rat where an iv infusion to information are orthogonal parameters and are both
steady-state was performed. A dose of 1 mg/kg was administered in required to truly appreciate brain distribution, particularly
all cases
for PB-PK approaches [48]. Figure 3a, b show the use of
PBPK modelling to elucidate the interplay between PS and
moderately BBB permeable marker while diazepam is brain tissue distribution for clozapine and acetaminophen
employed as a measure of perfusion limited uptake. following iv infusion to steady-state and the measurement
Knowing the dose concentration (Cpf mg/mL) vascular of brain ECF via microdialysis. Blood kinetics, BBB
volume correction (Vi mg/g brain) and brain concentration uptake and fubrain were measured experimentally [47] and
(Cbr mg/g brain) at time t enables the intrinsic uptake fitted to a PBPK model assuming rapid distribution
(Kin mL/g brain/min) to be calculated. between brain ECF and tissue. Predicted ECF
concentrations for both clozapine and acetaminophen lie
Cbr ðtÞ
 Vi ¼ Kin t within two-fold of the measured values highlighting the
Cpf ðtÞ utility of this predictive approach. The respective Kp, PS
Kin can be viewed as a clearance and may be and fubrain values for clozapine (23, 1250 mL/h/kg and
transformed to a permeability surface product (PS) using 0.01) and acetaminophen (0.67, 25 mL/h/kg and 0.72) were
the Crone–Renkin equation, where F represents flow (e.g. also very different but both attained steady-state ECF
the Kin of diazepam) [5]. concentrations in a similar time window of 2 and 4 h. An
  important observation is the concept of the intrinsic brain
Kin half-life, which has been defined as the time taken to reach
PS ¼ F  ln 1 
F 50 % of the steady-state Kp, and is inversely proportional

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to the hybrid term PSfubrain [48, 49]. If the product of PS
and fubrain is comparable between two compounds then
their time to attain brain equilibrium will be similar even if
they possess broadly different physicochemical properties,
as is the case for clozapine and acetaminophen showed in
Fig. 3 but also for other compounds where the observation
was made previously but the conection between PS and
fubrain had not been established [50].
All of this work is only really performed to better
understand the likely brain distribution of drug candidates
in humans. In vivo measurements in humans are largely
achieved through imaging studies (e.g. PET) but some
Fig. 2 Kin derived from Ref. a
[47]. Compounds are ordered
left to right in terms of
a increasing Kp and b increasing
Kp,uu (demonstrating no
correlation of Kin to Kp or Kp,uu)
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information is also available from post-mortem dissections
where brain tissue concentrations has been collected for
toxicology purposes [51]. On the limited data available
human brain distribution and in vitro brain tissue binding
measurements have shown reasonable correlations and
supports the view that passive permeability across the BBB
is important, at least for the compounds tested thus far [52].
Notably, there is a contention that passive permeability is
the exception rather than the norm as stated by Kell et al.
[53]. However, this argument neglects to consider the
influence of tissue binding as and the argument proposed
by Kell et al. has also been refuted in a recent repost [54].
J Pharmacokinet Pharmacodyn (2013) 40:301–314 307

a 0.14 tolerance, low cost and flexibility in adapting the lipid

membrane composition, the parallel artificial permeability
0.12
assay (PAMPA) has demonstrated reasonable correlations
0.10 to other cell-based models and in vivo evaluations in
rodents. By adapting a porcine polar brain lipid to mimic
ECF (uM)

0.08
the BBB membrane characteristics, PAMPA has also been
0.06
reported to display good correlations to in situ brain per-
0.04 fusion measurements [16]. This technique has been utilized
0.02
to provide early classification of test compounds into those
likely to be CNS penetrant and non-CNS penetrant, based
0.00
0 1 2 3 4 5 6 on permeability. However, because of the lack of active
Time (h) transporter expression or metabolic enzymatic activity,
b 7.0
PAMPA is limited in its application to generating the
passive permeability component of BBB uptake [6]. Sim-
6.0
ilarly, immobilized artificial membrane (IAM) column,
5.0 which contains stationary phase consisting of lipid mono-
ECF (uM)

4.0 layer, allows drug partitioning during chromatographic

3.0
separation. The greater retention of the column reflects
higher passive permeability although it suffers again from
2.0
the same limitation in mimicking only passive diffusion
1.0 [17].
0.0
0 1 2 3 4 5 6
Cell based in vitro methods
Time (h)

Fig. 3 Concentration-time profile of brain ECF following iv infusion
to steady-state for a clozapine and b acetaminophen. Measured brain
ECF concentrations (filled square) and predicted brain ECF concen-
trations (open square) are shown. Clozapine is characterised by PS
and fubrain values of 1250 mL/h/kg and 0.01, respectively. Acetami-
nophen is characterised PS and fubrain values of 25 mL/h/kg and 0.72,
respectively. Despite significant differences in the absolute values of
PS and fubrain between clozapine and acetaminophen the hybrid term
PSfubrain are comparable, leading to comparable brain equilibration
half-lives in vivo [48, 49]
In summary, there are a range of in vivo techniques
available to characterise the various aspects of the rate and
extent of brain distribution and the ensuing information is
amenable to providing accurate and predictive PBPK
models. However, the flip side of performing in vivo
experiment is the time required for the surgical procedures,
particularly for brain microdialysis and in situ brain per-
fusion. This has continued to drive the development of
in vitro based approaches with a view to initially triaging
the extensive number of molecules being synthesised dur-
ing target validation and lead optimisation.
In vitro methodologies
Non-cell based in vitro models
Early in vitro screening in Drug Discovery is mainly based
on the use of techniques that act as a surrogate measure for
transfer across the BBB. With high throughput, pH
Cell-based in vitro approaches to measuring permeability
provide a more physiologically relevant screening para-
digm over PAMPA and IAM columns. The experimental
design comprises two compartments that make contact
through a confluent cell membrane that displays a tight-
junction morphology. Drug in physiological buffer is
placed in the donor compartment and allowed to diffuse
across the cell layer for a given time period (t) after which
the concentration is determined from both the donor and
receiver compartments. If the cell boundary is polarised,
such as by the orientation of a transport protein then the
flux across that cell line must be measured in both direc-
tions. The apparent permeability (Papp) is the calculated as
shown below:
dCr Vr
Papp ¼ 
dt A  Cr ð0Þ
where Cr, Vr and A denote the receiver drug concentration,
volume of the receiver compartment and the area of the cell
membrane. Correcting for losses and adsorption can be also
accommodated through the use of Pexact values [55].
Caco-2 cells are a well established in vitro tool for
assessing intestinal absorption. The cell line derived from
human epithelial colorectal adenocarcinoma cells has been
adapted to evaluate BBB permeability [56]. Due to its non-
brain originated epithelial nature, several studies suggested
the limited utility of Caco-2 for assessing CNS penetration
[57]. Following this, the Madin–Darby Canine Kidney
(MDCK) cell line has been widely used as an in vitro

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permeability model due to the tighter monolayer junctions
achievable and the relatively short cell culture time
[15, 58]. Despite its non-human cerebral and epithelial
origin, MDCK has been stably transfected by a retrovirus
carrying the multi drug resistance (MDR1) gene in order to
incorporate P-gp, which is the most prolific efflux trans-
porter of potential CNS drugs in the BBB. In a comparative
study of several different P-gp assay formats the MDR1–
MDCK assays faired favourably against the comparators
[59]. Furthermore this study also showed a strong corre-
lation between the in vitro efflux ratio between MDCK cell
lines transfected with the rodent mdr1a gene and human
MDR1 gene. Although P-gp is one of the most important
efflux transporters and its role is vital in restricting the
brain penetration of many exogenous substances, active
transport of drugs from the brain is not limited to this efflux
pump alone [60–62]. BCRP, another predominant efflux
protein in human brain, has been the subject of emerging
attention although its impact remains controversial [63].
This transporter plays a dominant role in limiting sorafenib
transport to the brain in vivo, which was clearly demon-
strated using the BCRP gene stably expressed into the
MDCK cell line [64]. There is no doubt that significant
advantages are there for in vitro cell lines that express
multiple active transporters in order to provide a more
comprehensive and cost effective evaluation of efflux lia-
bilities. Recently, Poller et al. reported a study using a
double-transduced, polarized MDCKII cell line expressing
both human P-gp and BCRP in order to assess the possible
interplay between these two efflux proteins for drug
transportation across BBB [65].
The next tier of in vitro permeability models involve
the use of BBB cell cultures and originated in the 1990s
[66, 67]. Primary cultured brain endothelial cells have been
isolated and reported from several species including
bovine, porcine, rat and mouse brain [68–71]. In a com-
parative study, a bovine brain microvessel endothelial cell
model demonstrated a superior correlation to in vivo brain
microdialysis results relative to Caco-2 and MDCK/MDR1
data for a series of nine reference compounds after exclu-
sion of P-gp substrates [56]. A tantalising refinement of this
approach involves the use of human microvascular endo-
thelial cells co-cultured with various concoctions of
astrocytes, glial cells and pericytes [72, 73]. The predictive
value of humanized in vitro models has been demon-
strated in combination with clinical (PET) [57]. However,
despite the intensive effort in this field, the majority of
these immortalized brain endothelial culture models
struggle to achieve a sufficient tight junction morphology
and to maintain a full complement of transporter func-
tion or enzymatic activity that really make them most
appealing.
123
J Pharmacokinet Pharmacodyn (2013) 40:301–314
In vitro assessment for CNS delivery (extent and brain
distribution)
It is widely accepted that unbound drug concentrations are
more relevant to understanding pharmacodynamic efficacy
because this is the portion of drug available to bathe the
receptor [74, 75]. The extent of drug biodistribution into
the brain has been the subject of increasing attention and is
largely governed by binding to blood constituents and to
brain tissue [76]. For passively permeable compounds good
in vivo–in vitro correlations have been established in
rodents [30], pigs [45] and humans [52]. This is because
Kp,uu is equal to unity for passively permeable compounds
and hence Kp is corrected by the relative tissue binding as
shown below:
   
Cubrain Cbrain fubrain
Kp;uu ¼ ¼ 
Cubloodorplasma  Cblood or plasma fublood or plasma
fubrain
¼ Kp  :
fublood or plasma
Unbound fractions should not be used by themselves to
guide the selection of compounds within Drug Discovery
because they merely represent the proportion of available
drug but not the actual unbound concentration. The
unbound fraction in blood, plasma or brain should be
integrated and interpreted with total concentrations or
in vivo exposures [77]. Generally unbound brain fractions
are generated by means of tissue homogenates but evidently
the entire cellular structure of the brain parenchyma is lost
during this preparation [74, 76]. An alternative approach is
to employ fresh in vitro brain slices, which have been shown
to retain at least some transporter activity as well as cell
morphology [11]. A thin slice of brain is bathed in
simulated ECF containing the drug after which the tissue
slice is homogenised and analysed by means of LC–MS/MS
analysis. This method yields an unbound brain volume of
distribution (Vu,brain) for the drug (as opposed to fubrain) that
is derived from the following equation shown below where
Abrain denotes the amount of drug in brain slice and Cubrain
is the concentration of free drug in the incubation solution:
Abrain
Vu;brain ¼
Cubrain
The advantage of the brain slice method lies in the fact
that cellular barriers, cellular pH gradients and also efflux/
influx transporters are retained in addition to tissue binding
component [76]. As Vu,brain is an apparent volume term, it
reflects the volume of distribution into the brain tissue
relative to the ECF volume (0.2 mL/g) [11, 76]. Values
close to 0.2 mL/g indicate little or partial distribution into
the brain parenchyma whereas progressively higher values
(e.g. [0.8 mL/g) indicate substantial nonspecific binding
J Pharmacokinet Pharmacodyn (2013) 40:301–314
into brain tissue. The use of brain tissue slices is more
involved than the preparation of homogenates but it is clear
that slices offer a closer resemblance to the brain in vivo
and is likely to introduce fewer artefacts as a result [11].
In silico methodologies
In silico models are available for the estimation of several
brain-related parameters including PS [78, 79], Kp (e.g.
expressed in logarithmic form and denoted as logBB)
[80, 81] fubrain [82], Kp,uu [83] and P-gp efflux [84, 85].
Evidently the value of these tools is for early Drug Dis-
covery where there is often a need to triage a large numbers
of compounds. Of these in silico tools there is substantial
literature for logBB, which continues to appear despite an
accumulating body of evidence to suggest that this
parameter is of little value in selecting or triaging com-
pounds [27, 75]. In addition there is a general miscon-
ception that logBB is related to BBB permeability [80, 81]
whereas it strictly refers to partitioning. Kp and its in silico
counterpart logBB are complex hybrid parameters because
they incorporate the relative extents of tissue binding to
brain and blood (or plasma) plus the concentration gradient
across the BBB caused by active transport. Pharmacolog-
ical effect is not driven by the total concentration as
unbound drug is the portion that is available to bind to the
targeted receptors [27]. An excellent example of this was
cited by Watson et al. [77] where the in vivo D2 receptor
occupancy in rats was best correlated to Cubrain/Ki(D2) or
more explicitly the unbound brain concentration relative to
the drug’s in vitro potency at the D2 receptor. Risperidone
was the least brain penetrant in terms of Kp but was offset
by the fact it possessed the highest unbound brain con-
centration married to its high D2 potency. So logBB is
useful in the sense that it can predict brain distribution but
the absolute value is meaningless as is also the case for the
in vivo measurement of Kp.
Due to the introduction of in vitro brain tissue binding
screens, in silico models predicting fubrain values have been
reported in the literature [82]. Table 4 highlights the
composition of the brain relative to plasma and other tis-
sues. Based on the high lipid content of the brain it is not
surprising that fubrain is inversely proportional to logP
[9, 82] although this accounts for only around 50 % of the
overall variance. Incorporating a wider range of molecular
characteristics into an in silico model markedly improves
the correlation [82] but it is important to remember that
fubrain data is still largely derived from brain homogenates
rather than brain slices. Substantial discrepancies have
been found between these two measurements [76] and have
been attributed to loss of pH differences across cellular
barriers and also the loss of latent transporter activity that is
309
present in slices but not homogenised tissue. In this latter
work Frı́den et al. [76] proposed a correction for fubrain to
account for these differences, but inevitably, in silico
approaches based on brain slice data would be the best
option.
Estimating PS values in silico has been facilitated by the
widespread use of control compounds when conducting
these experiments. Once again lipophilicity is an important
component of the model but other physiochemical factors
modulate this. Early literature reports from in situ brain
perfusion studies had showed a strong correlation between
increasing BBB uptake and increasing logP [5]. However,
the range of compounds tested were characterised by logP
values \1 and were not typical of the chemical space
generally occupied by CNS drugs [47]. In addition BBB
uptake cannot increase indefinitely and would become
limited ultimately by the perfusion flow rate [6, 87]. Fig-
ure 2 shows that many common marketed CNS drugs are
characterised by comparable Kin values to that of diaze-
pam, the common marker of perfusion limited uptake.
Indeed some agents such as sertraline and maprotiline are
characterised by Kin values much greater than would be
expected from the traditional diazepam standard [7, 47, 87].
Active transport also influences BBB uptake and the
number of examples collated from in situ perfusion studies
has increased markedly [5, 6]. This strikes at the heart of
many in silico models for estimating brain distribution.
Essentially any global model has to contend with the
structure activity relationships (SAR) of all BBB trans-
porter interactions with drug molecules. This is highly
problematic because the SAR associated with the substrate
specificity for transporting gabapentin [88] or other sys-
tems [35–41] would differ greatly from that of P-gp
substrates.
P-gp occupies a particular level notoriety within CNS
Drug Discovery because of its highly promiscuous sub-
strate specificity. P-gp substrate models have used a range
Table 4 Compositions of various human organs as weight fractions
of lipid, protein and water showing the higher lipid content of brain
with respect to blood [86]
% Weight fractions
Tissue Lipid Protein Water
Blood 0.65 18 80
Brain 10.7 7.9 79
Fat 80 5 15
Liver 7 18 72
Kidney 5 17 77
Muscle 2 17 79
Lung 1 17.7 78
Heart 10 16.7 72.7
123
310 J Pharmacokinet Pharmacodyn (2013) 40:301–314

Table 5 Brain distribution in rodents for a series of marketed drugs et al., investigated the brain distribution of acetaminophen
measured in terms of Kp for wild-type (WT) mice and mdr1a/b implanting microdialysis probes in the striatum to measure
knockout (KO) mice and Kin
brain ECF concentrations in rats, and in lateral ventricle
Rodent Kp WT Kp KO Kp ratio and cisterna magna for sampling CSF concentrations. This
Kin miceb miceb work was able to characterise the differences between the
(mL/min/g) mdr1/b mdr1/b (KO/WT)
(?/?) (-/-)
brain ECF and CSF concentration-time profiles in rodents
but then allowed the human brain ECF concentrations to be
Quinidine 0.0299e 0.28 10.07 36.5 predicted based on human CSF data by extrapolation.
e
Verapamil 0.299 0.34 5.63 16.5 Several examples of PBPK predictions of drug pharma-
Risperidone 0.849a 0.78 8.02 10.3 cokinetics in humans and drug–drug interaction have been
Metoclopramide 0.125a 1.22 7.86 6.5 accepted by regulatory agencies, so PBPK modelling and
Sumatriptan 0.00120a 0.02 0.09 3.7 its uses in supporting the filing of therapeutic CNS treat-
Digoxin 0.00197d 0.05 1.4 28.0 ments is expected to evolve in the future.
Loperamide 0.0986c 0.08 2.61 32.6
Risperidone 0.849a 0.44 5.52 12.7
Morphine 0.0104e 0.53 1.51 2.9 Conclusions
Metaclopramide 0.125a 0.79 5.48 6.9
Mesorizadine 0.837a 0.95 16.90 17.8 Distribution of drugs from the vascular compartment into
Thiothixene 2.81a 2.26 24.63 10.9 the brain is governed by both the rate of passage of
Citalopram 0.580a 5.72 17.87 3.1 unbound drug across the BBB and the extent of distribution
a in brain tissue across the intervening cellular barriers.
Taken from Ref. [47]
b Mechanisms for passive diffusion, active transport, tissue
Steady-state measurements in house
c
binding and pharmacokinetics work in concert to produce
Taken from Ref. [95]
d
the concentration-time profile observed in vivo. The range
Taken from Ref. [46]
e
of in vivo, in vitro and in silico tools has increased
Taken from Ref. [96]
markedly over recent years and have contributed to the
improved understanding of the underlying mechanisms
of novel methods to contend with this varied SAR associated with drug distribution into the brain and also in
including more complex machine learning approaches [89]. triaging compounds from new chemical templates that
However, being a P-gp substrate does not necessarily show potency in vitro. The over interpretation of in vivo Kp
prevent in vivo distribution to the brain as shown in measurements or in silico logBB values is now less com-
Table 5 for a series of marketed CNS drugs. This is mon as it much more widely appreciated that unbound drug
because the unbound concentration of drug available to concentrations are more relevant with respect to both
cross the BBB from blood together with the corresponding efficacy and brain distribution. Measurement of in vitro
PS may be sufficient to partially offset the active efflux free fractions in brain and blood (or plasma) have enabled
process and hence still afford the attainment of efficacious Kp values to be converted to Kp,uu in an attempt to
concentrations within the brain. understand the mechanisms associated with BBB passage
PBPK modelling is another in silico approach that in vivo. Assessing BBB permeability remains one of the
merges compound specific parameters (e.g. PS, fubrain) and early interventions in the lead optimisation process to tri-
system specific parameters (e.g. organ blood flow) using a age compounds that display inherently poor physico-
mathematical description of the body in order to predict PK chemical properties or those subject to P-gp efflux, or
parameters [21]. Other in vitro data, such as drug perme- perhaps BCRP efflux.
ability, may also be incorporated into whole body PBPK Literature reports looking at marketed CNS drugs have
models in order to improve their predictive power as shown shown broadly comparable findings in that many appear to
for morphine and oxycodone [90]. Contribution of active be the subject to passive diffusion with a small portion
efflux for morphine was obtained using an in vitro Caco-2 substrates for P-gp but still attain sufficient albeit reduced
cell model whilst active influx contribution to oxycodone brain concentrations. The reality of CNS Drug Discovery is
was evaluated by immortalized rat brain capillary cells. somewhat more complex however because a far wider
The incorporation of these in vitro input parameters rea- variety of chemical templates are being tested and so the
lised a significant improvement in the prediction of the chances of mismatches between in vivo and the current
unbound drug concentration-time profile. Another advan- in vitro measurements increase. This is unsurprising from
tage of PBPK approaches is the capability to determine the perspective of drug transport because contemporary
regional differences within the brain [21, 92]. Westerhout CNS drug screening has not moved much further than P-gp

123
J Pharmacokinet Pharmacodyn (2013) 40:301–314
and because the expression of native transporters on cell
lines such as MDCK is not well understood. If Kp,uu is
markedly lower than unity whereas in contrast in vitro
assessment indicates no likely P-gp efflux then there are
still few experimental options to resort to. From the clinical
perspective it still remains difficult to extrapolate P-gp data
across species and it is unclear whether drug–drug inter-
actions are likely in humans [92].
BBB models based on primary brain cultures would
afford a better tool for screening out compounds across a
wider range of transporters but few Drug Discovery groups
have invested the time needed to make this a standard and
less tedious approach. Indeed one of the biggest issues with
in vitro permeability tools is the generally poor correlation
of values between laboratories running seemingly similar
screens even for simpler models based on MDR–MDCK
[93]. Although these instances are not in the majority it
does become very challenging to guide the medicinal
chemistry effort from a CNS DMPK perspective and
arguably leads to more compounds being tested in vivo
because of the inability to triage compounds effectively.
This also appears to be genuine problem and not an artefact
because the predictive power of PBPK modelling is
excellent when in vivo measurements are made such as PS
or brain ECF concentrations. So there remains an inherent
tension in the Drug Discovery paradigm between in vitro
models that afford adequately high throughput versus the
need to provide better translational data so that the right
molecules are identified and progressed more rapidly [94].
Perhaps the compromise lies in some form of the brain
slice methods [11] and the in silico counterparts [83]
because these assays retain cellular characteristics yet are
simpler than those based brain cell cultures.
In summary our understanding of brain distribution has
been improved greatly by the range of newer in vivo,
in vitro and in silico approaches and particularly those that
provide information on unbound concentrations or
unbound partitioning. Long term this can only lead to the
selection of better CNS drug candidates and ideally assist
in improving the high attrition rates that afflict this field of
pharmaceutical research.
Acknowledgments The authors would like to thank Dr. Andy
Ayrton for his valuable scientific input during the preparation of this
manuscript.
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