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Current Medicinal Chemistry, 2016, 23, 1392-1407
eISSN: 1875-533X ISSN: 0929-8673

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Selecting Good ‘Drug-Like’ Properties to Optimize Small

The
International
Journal for
Timely In-depth
Reviews
in Medicinal

Molecule Blood-Brain Barrier Penetration Chemistry

BENTHAM
SCIENCE

Paul C. Trippier*

Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health

Sciences Center, Amarillo, TX, USA and Center for Chemical Biology, Department of Chem-
istry and Biochemistry, Texas Tech University, Lubbock, TX, USA
Please provide
corresponding author(s)
Abstract: The success rate to achieve clinical approval of drugs developed to treat diseases photograph

of the central nervous system (CNS) is the lowest of all disease indications. A large contribu-
tor to this poor success rate is failure of small molecules to pass through the blood-brain bar-
rier (BBB), a barrier composed of capillary endothelial cells connected by tight junctions
that functions to extrude xenobiotics from the brain. Designing small molecules to be BBB
penetrant has been the subject of intensive research and has resulted in a series of guidelines to attain the best
 
 

possible chances of BBB penetration. This review will analyze the current state of thinking in ranking the im-
portance of various physicochemical properties required to select BBB penetrant molecules, describe model
systems to determine BBB penetration, summarize data analysis methods and provide an outlook on further
developments in the field.
Keywords: Blood-brain barrier, Drug-likeness, Central Nervous System, Neuropharmacology, Physicochemical
Properties, Drug Design.
Received: April 04, 2015 Revised: March 22, 2016 Accepted: April 04, 2016

INTRODUCTION packed monolayer lining all of the surfaces of the cap-

illaries supplying essential nutrients to the brain [4].
The development of new therapeutics to treat cen-
CNS targeting NCEs must penetrate through this bar-
tral nervous system (CNS) disorders and diseases suf-
rier before they can enter the brain and interact with
fers from the lowest statistical chance of success of all
their therapeutic target. This transport is predominantly
drug categories. The length of time required to bring a
by transcellular passive diffusion, a process that is in-
CNS indication drug to market is estimated at 12-16
fluenced by the same physicochemical properties as for
years, compared to 10-12 years for other disease states
transport across cellular membranes and within the gas-
[1]. Off the total number of new chemical entities
trointestinal (GI) tract, albeit, with much stricter toler-
(NCEs) that enter phase I clinical trial for CNS indica-
ances.
tions only 8% proceed to clinical approval [2]. In order
for such NCEs to penetrate the brain and engage their The existence within the BBB of efflux transporters
target they must pass through the blood-brain barrier serves to further limit total brain penetration for many
(BBB), a feat estimated to be unachievable by 98% of compounds [5]. Of all the active efflux transporters
potential drug molecules [3]. present in the BBB P-glycoprotein (P-gp) is perhaps
the most adversarial in extruding NCEs from the brain
The BBB is more limiting to compound permeabil-
before they can engage their target of action [6-8]. This
ity than most other barriers within the body as it is re-
has led to many efforts to design predictive models to
sponsible for ensuring homeostasis of the brain by pro-
assess substrates of P-gp, prior to the synthesis of com-
tecting against the incursion of toxic substances. It is
pounds for biological evaluation [9]. However, recent
composed of endothelial cells that form a tightly
years have seen the identification of additional active
*Address correspondence to this author at the School of Pharmacy,
efflux transporters at the BBB. While these transporters
Texas Tech University Health Sciences Center, Department of are not as limiting to brain penetration as P-gp they do
Pharmaceutical Sciences, 1300 S. Coulter, Amarillo, TX 79106, represent a non-trivial effect on overall penetration to
USA; Tel: 806-414-9245; Fax: 806-356-4034; the brain [10].
E-mail: paul.trippier@ttuhsc.edu

1875-533X/16 $58.00+.00 © 2016 Bentham Science Publishers

Selecting Good ‘Drug-Like’ Properties to Optimize Small Molecule
While efflux transporters at the BBB represent a
significant challenge for penetration, influx transporters
offer an opportunity to enhance penetration. These
transporters facilitate the uptake of essential molecules
(such as amino acids and peptides) from the blood that
would otherwise be excluded from the brain [11]. The
design of CNS drugs to act as substrates of influx
transporters is extremely difficult with most successful
substrates discovered through serendipity; almost all
are amino acid-like structures. However, the large
amino acid transporter 1 has been shown to be suscep-
tible to recognizing small molecules by use of a pro-
drug strategy, indicating that it may one day be possi-
ble to target influx transporters as a mechanism to cross
the BBB [12]. Currently, influx transporters at the BBB
[13], such as the organic anion transporting polypep-
tides (OATP) play a role in BBB penetration of BBB
permeable therapeutics [14]. For a comprehensive re-
view on the subject of transporters at the BBB the
reader is directed to a recent article [15].
Another factor (albeit a contentious one) to consider
in penetrating the BBB is plasma protein binding where
opinion of the importance of this facet of drug design is
split [16-18]. Structural and physicochemical properties
that make small molecules effective drugs can also
make them effective protein binders. NCEs that dem-
onstrate reduced penetration over predicted values may
be exhibiting high levels of plasma protein binding in
the circulation.
A critical consideration in designing compounds to
penetrate the BBB is that its function can be effected
by disease pathology [19]. Changes that can occur at
the BBB in several CNS conditions include altered ex-
pression levels of transporters in the endothial cells
which comprise the BBB, and increased ‘leakage’
through tight junctions [18, 20, 21]. This changing
facet of the BBB makes prediction of penetration even
more fraught, especially when one considers an aging
population and the often complicated multiple health
conditions that this population displays.
This review article will address the current state of
the field in what constitutes good ‘drug-like’ properties
to enable BBB penetration. Physicochemical properties
will be examined as to their relationship to BBB pene-
tration. Model systems for predicting penetration, both
physical and virtual, will be summarized and the suc-
cess of these models and guidelines will be assessed,
with future directions addressed.
At the outset of this review it is critical to define the
terms ‘permeability’ ‘distribution’ and ‘penetration’. It
is often the case that many literature reports inter-
Current Medicinal Chemistry, 2016, Vol. 23, No. 14 1393
change the terms permeability, penetration, crossing,
transport across, etc. Is this correct or does each term
have subtle differences?
BBB Permeability (Papp)
This term is defined as the rate at which the com-
pound in question crosses the BBB [22]. When used as
a sole definer for evaluation of a compound, care
should be exercised and the nature of the disease/target
evaluated. If rapid permeability is required for thera-
peutic onset, a low Papp represents a legitimate concern
about the potential efficacy of the NCE. However, if a
compound is designed to counter a disease that requires
a steady release of the drug into the brain (e.g. depres-
sion) a low Papp is acceptable, albeit not too low as to
render insufficient levels of the drug into the brain. As
a general measure of potential for a compound to cross
the BBB, without considering physiological effects,
Papp is an excellent parameter allowing direct compari-
son between NCEs.
Brain Distribution
This term can be defined as the concentration of un-
bound drug in the brain (Cb,u) versus the concentration
of unbound drug in the plasma (Cp,u), Cb,u/Cp,u or
Kp,uu,[23] (also called Kp,free) [24]. A compound of in-
terest will distribute between the plasma and brain at a
rate determined by its permeability. Compounds with
high plasma protein binding may experience low rates
of permeability and result in poor BBB penetration no
mater how high their Papp [16]. However, other theories
posit that high plasma protein binding will have no ef-
fect on BBB penetration [17]. The BBB distribution is
in effect a measure of quantity (or extent) of compound
crossing the BBB. A Kp,uu close to 1 represents an equi-
librium of the compound between plasma and brain. By
measuring plasma concentration Kp,uu takes into ac-
count the effect of permeability and efflux transporters
[23, 25]. One way to increase the unbound drug con-
centration in the brain is to increase the dose of com-
pound given, which raises unbound concentration in
the plasma.
BBB Penetration
A term encompassing both distribution rates and
permeability representing an overview of how well a
compound will cross the BBB. There is no overall dif-
ference between BBB penetration, BBB permeability,
or brain distribution.
Many additional parameters have been defined to
help the drug discovery process predict and assess BBB
1394 Current Medicinal Chemistry, 2016, Vol. 23, No. 14
penetration such as total brain to plasma ratio (B/P),
efflux ratio (ER) and fraction of drug unbound (fu,p or
fu,b). The reader is directed to an excellent review that
discusses the relative benefits and reliability of these
factors to CNS drug discovery [22]. In this review we
will consider mainly those factors affecting BBB per-
meability and their implications to overall BBB pene-
tration.
PHYSICOCHEMICAL PROPERTIES INFLU-
ENCING BBB PENETRATION
The physicochemical properties of a molecule
are directly related to its ability to cross the BBB [26].
To the medicinal chemist this represents both a signifi-
cant opportunity to tailor the structure of compounds to
increase the chances of constructing a successful CNS
therapeutic, and a considerable challenge to ensure a
NCE retains ability to engage its target.
The ‘standard’ predictor of ‘drug-likeness’ for
membrane permeation and absorption are Lipinski’s
rule of five that apply only to orally administered drugs
[27]. These rules were derived after analysis of >2000
clinically approved and late stage clinical trial drug
candidates and apply only to drugs that absorb by pas-
sive diffusion through cell membranes. Those com-
pounds that are substrates for transporters are excep-
tions to the rule. The rule states that to cross a cell
membrane a compound should meet defined criteria in
molecular weight, lipophilicity, number of hydrogen
bond donors, and number of hydrogen bond acceptors.
As noted above, however, the BBB is not just any cell
membrane and it was soon discovered that Lipinski’s
original rules required modification to be applicable to
BBB penetration.
Analysis of clinically effective CNS drugs revealed
that compared to other indication drugs the CNS drugs
possessed lower molecular weight, lower polar surface
area (PSA, a measure of overall charge on the mole-
cule), fewer hydrogen bond donors, higher lipophilic-
ity, and were overall more rigid [26, 28]. Of these
properties it was classically regarded that lipophilicity
was key. A general linear relationship between lipo-
philicity and BBB penetration was believed to exist in
that the higher the lipophilicity of a compound the
greater its penetration through the BBB [29-31]. Lipo-
philic regions of molecules do not contain functionali-
ties capable of forming hydrogen bonds, hence they are
not soluble in water. This relationship between hydro-
gen bonding groups and lipophilic groups has a long-
standing importance in CNS drug design. However, as
Paul C. Trippier
discussed later, current trends are moving away from
this classical view [32].
A revised set of rules [33] for the design of success-
ful BBB permeable compounds stated that compounds
should contain:
• <8-10 functional groups capable of forming
hydrogen bonds
• A total molecular weight limit of 500 with
<400 preferred
• No acidic functional groups
Over time these rules have been refined as more ac-
curate model systems for assessing BBB penetration
have become available [32, 34], greater numbers of
successful CNS drugs have been clinically approved,
and their structural features revealed. This information
has led to a general consensus that hydrogen bond do-
nors are more limiting to BBB permeability than hy-
drogen bond acceptors. When one considers the chemi-
cal composition of phospholipids that comprise the
BBB (Fig. 1) it is not surprising that hydrogen bond
donors limit compound penetration. Phospholipids
have a polar head group comprised of nitrogen, oxygen
and phosphorous moieties that are hydrophilic and two
hydrophobic hydrocarbon tails. Any NCE with avail-
able hydrogen bond donors that comes into proximity
with the polar head group will interact and form hydro-
gen bonds with the multiple electronegative atoms (hy-
drogen bond acceptors), thereby slowing or halting its
progress through the BBB. As the phospholipid con-
tains no hydrogen bond donors, a compound with only
hydrogen bond acceptors will not interact and will pass
by the polar head at a faster rate.
To account for these observations a new set of rules
for penetrating the BBB where proposed [35, 36]. In
order to penetrate the BBB a compound should have
the following characteristics:
• Total number of nitrogen and oxygen atoms
(N+O) <6
• Polar surface area <60-70 Å2
• Molecular weight <450
• Log D (distribution coefficient, a measure of
total lipophilicity of ionized and unionized
compound) = 1-3
• Clog P (partition coefficient, a measure of
lipophilicity of the unionized compound) –
(N+O) = >0
Selecting Good ‘Drug-Like’ Properties to Optimize Small Molecule Current Medicinal Chemistry, 2016, Vol. 23, No. 14 1395

Fig. (1). A): Structure of the representative BBB phospholipid phosphatidylcholine illustrating the polar head and hydrophobic
tails. Top: chemical structure. Bottom: Ball and Stick model. B): Drug molecules with few or no hydrogen bond donors, only
hydrogen bond acceptors will not bind to the phospholipid and will pass through the BBB at a faster rate. C): Drug molecules
with large numbers of hydrogen bond donors will form hydrogen bonds (dashed lines) to the hydrogen bond acceptors on the
phospholipids of the BBB and will be slowed or stopped from passing through the BBB.

An amendment to Lipinski’s original rules was pro-
posed by Veber et. al. after analyzing 1100 orally
bioavailable drugs [37]. They concluded that reduction
of PSA correlates better to increased cell penetration
than does lipophilicty. The defining factor in reducing
PSA was attributed to the number of rotatable bonds in
the molecule – the more rotatable bonds, the higher the
PSA value and the lower the penetration rate. It was
established that >10 rotatable bonds reduces oral
bioavailability. Penetration through cellular membranes
is analogous with BBB penetration (albeit the BBB is
exceedingly more difficult to penetrate) and most CNS
drugs have significantly higher rigidity than other
classes [26], for example the Alzheimer’s Disease drug
memantine compared with the antiviral Acyclovir (Fig.
2). Most centrally acting drug compounds have less
than five rotatable bonds [38].
Fig. (2). Chemical structures of the highly rigid clinically
available Alzheimer’s Disease drug Memantine and the
flexible clinical antiviral drug Acyclovir.
In addition to these rules it was noted that acidic
functional groups correlated with poor BBB penetra-
tion. Analysis of the structure of clinically successful
CNS drugs reveals that approximately 75% are basic,
19% are neutral and only 6% are acidic [39]. Basic
drugs at physiological pH are largely cationic and can
interact with the charged phospholipid heads, facilitat-
ing passive diffusion [40]. This is a rather fortuitous
happenstance as the majority of CNS drugs rely on ba-
sic functionality in their pharmacophore to exhibit ac-
tivity, similar to the endogenous monamine neuro-
transmitters, a popular scaffold for CNS drug discov-
ery. The same functionality it would appear, also as-
sists in BBB penetration.
It is evident that all of these parameters define, to
some extent, the overall lipophilicity of a drug candi-
date. Electronegative (nitrogen and oxygen) and ionic
species (defined to an extent by PSA) are capable of
forming hydrogen bonds and therefore are hydrophilic
(lipophobic) [41]. Optimum levels of lipophilicity for
BBB penetration have been reported to be representa-
tive of a logD = 2-3 [42]. Is lipophilicity therefore the
most important aspect for the medicinal chemist to
consider when designing a new CNS therapeutic? Re-
cent studies based on new experimental methods to
determine Kp,uu (see model section) have revealed that
unbound drug concentration in the brain does not corre-
late to lipophilicity (as was suggested by a previous
study measuring total brain-to-plasma ratio) [32]. A
clear dependence of BBB penetration on hydrogen
bonding was demonstrated. This was attributed to the
‘additive effect of reducing passive permeability while
increasing the possibility of interactions with efflux
transporters’. The study demonstrates that the addition
of two hydrogen-bond acceptors to a CNS active drug
results in a doubling of its therapeutic window. This
1396 Current Medicinal Chemistry, 2016, Vol. 23, No. 14
again reiterates the importance of avoiding hydrogen-
bond donors to enhance BBB penetration.
IN SILICO MODELS OF BBB PENETRATION
With the advent of more powerful and accurate
computer software for simulating (bio)chemical proc-
esses significant effort has been invested in predictive
models of BBB penetration [43-46]. Prediction of BBB
penetration is a complex and challenging area when
one considers the multitude of factors requiring simula-
tion – uptake, plasma protein binding, efflux pumps,
influx transporters, brain tissue binding, first-pass
metabolic clearance, etc. However accurate prediction
software is, the drug molecule under analysis must ul-
timately be synthesized and tested for BBB penetration
in in vivo and/or in vitro model systems. The use of in
silico prediction provides a relatively inexpensive
(compared with animal models) and rapid initial screen
for BBB penetration capability of screening libraries.
Perhaps the first and most widely used in silico pre-
diction of BBB penetration is that of lipophilicity cal-
culation – calculated logP (ClogP) [47]. While calcula-
tion of this parameter is now a standard feature in a
variety of widely used chemical drawing packages its
value is limited. Lipophilicity is only one physico-
chemical aspect of a compound that influences BBB
penetration and, as discussed above, is now considered
less of a priority than hydrogen bonding when consid-
ering structural modifications to drug candidates. A
recent analysis of 119 marketed CNS drugs and Pfizer
CNS preclinical candidates indicated that higher lipo-
philicity (ClogP >3) is detrimental to BBB penetration
and reduces toxicity potential [48].
The same group developed a CNS molecular pa-
rameter optimization (MPO) algorithm that may be
applicable to identifying drug candidates with an en-
hanced ability to become clinically approved CNS
drugs. The MPO approach considers six physicochemi-
cal properties of a compound under analysis; ClogP,
ClogD, molecular weight, topological PSA, number of
hydrogen bond donors, and most basic center (pKa).
Each parameter is assigned a desirability score ranging
from 0-1 with weighting for lipophilicity and pKa. The
algorithm was applied to a set of 119 marketed CNS
drugs as a test set. Results showed that 74% of these
successful CNS drugs had a high MPO score of ≥4
suggesting the value of this method in predicting suc-
cessful CNS drugs [49].
Many models have been developed that attempt to
classify compounds as either BBB+ (BBB permeable)
or BBB- (not BBB permeable) [50-52]. Indeed, a pre-
Paul C. Trippier
dictive in silico model was used to determine that
BBB+ compounds have a N+O value of <5 and ClogP
– (N+O) should be positive, which were incorporated
into the revised rules for BBB penetration presented
above [53]. A key consideration when evaluating the
usefulness of computer simulation software for assess-
ing BBB penetration is the training set of molecules
used to establish a baseline for BBB penetration. If the
molecule under consideration varies significantly in its
structure from the training set, prediction reliability and
accuracy is impeded. Currently in silico models have
approximately 80% predictive accuracy [54].
One of the most difficult challenges to in silico pre-
diction software is accounting for the effects of P-gp
(and other transporter) substrates. It has been calculated
that approx. 70% of clinical CNS drugs are highly
permeable and lack P-gp efflux, however, approx. 20%
of clinical CNS drugs show both low permeability and
are P-gp substrate specific [48]. No explanations have
yet accounted for this observation. As a result of this
situation and the lack of a known ‘pharmacophore’ for
P-gp substrate specificity (Fig. 3) few predictive mod-
els take into account the effects of P-gp efflux. In an
attempt to address this situation the Biopharmaceuticals
Drug Disposition Classification System (BDDCS),
which has been used to predict drug disposition in or-
gans throughout the human body was utilized as a
baseline dataset for predicting BBB penetration [55].
Upon combining BDDCS with in vitro and in silico P-
gp substrate data it was found that >90% prediction
accuracy could be achieved. Compounds that are pre-
dicted as class I drugs in BDDCS should be prioritized
for CNS indications [56].
While recent efforts are making advances in sub-
strate predictability they are hampered by the very na-
ture of the efflux transporter including the existence of
multiple binding sites dictating a much more promis-
cuous range of substrate possibilities [57]. An in silico
dynamic trans-membrane model of P-gp has recently
been reported that successfully predicted the substrate
binding residues of anticancer agents. The model was
constructed using homology modeling of human P-gp
based on the structure of C. elgans P-gp, molecular
docking, molecular dynamics and binding free energy
calculations [58]. Pharmacophore mapping of P-gp
substrates and non-substrates has led to the identifica-
tion of pharmacophoric moieties for substrate activity,
namely hydrogen bond acceptor, positive ionizable,
aromatic ring and hydrophobic groups [59]. These
pharmacophores were confirmed when docking was
performed into the P-gp binding cavity [60] of a newly
Selecting Good ‘Drug-Like’ Properties to Optimize Small Molecule
Fig. (3). Illustration of the wide chemical space occupied by three substrates of P-gp. The rigid cyclic structure of the H1 His-
tamine antagonist Loratidine, the flexible L-type calcium channel blocker Verapamil, and the steroidal glycoside cardiac drug
Digoxin. No pharmacophore for predicting P-gp substrate specificity has yet been determined.
resolved X-ray structure of murine P-gp [61]. Sepa-
rately a data set composed of 423 P-gp substrates and
399 non-substrates confirmed these findings. Compari-
son of eight physicochemical properties resulted in mo-
lecular weight and solubility being identifed as critical
for predicting P-gp substrates or non-substrates. This
training set lead to a predictive accuracy of 91.2%,
with a predictive accuracy of 83.5% for a test set of
200 compounds. Hydrogen bond acceptors in particular
spatial patterns along with flexibility were found to be
essential to distinguish P-gp substrate likeness [62].
Further, a set of 15 favorable and 15 unfavorable frag-
ments for the design of P-gp inhibitors has been identi-
fied using a training set of 973 compounds [63].
The process of quantitative structure-activity rela-
tionship (QSAR) has been used extensively for design-
ing CNS active drug candidates [64]. Traditionally this
process uses the classical Hansch algorithms to corre-
late observed biological activity to physicochemical
properties. Generally QSAR model systems have been
shown to have an average predictive performance of
within 0.4 log unit [51]. More recent models have ap-
plied BBB permeability-surface area coefficient data
combined with lipophilicty and hydrogen bonding pre-
dictors [65, 66] as well as statistical approaches using
molecular energy related discriptors combined with
lipophilicity [67]. For a more detailed review of QSAR
in CNS drug discovery the reader is directed to several
excellent reviews [26, 68, 69]. Computer prediction
software can narrow down a large library of com-
pounds to those most likely to penetrate the BBB but
ultimately the compound must be synthesized and its
ability to penetrate the BBB measured.
Current Medicinal Chemistry, 2016, Vol. 23, No. 14 1397
IN VITRO MEASUREMENT OF BBB PENETRA-
TION
Two concepts are important to the measurement of
brain penetration; rate and extent. The initial rate of
brain uptake is a measure of BBB permeability ex-
pressed, most often, as the permeability surface area
(PS) [70]. This measure has several advantages to
measure BBB penetration as it is not affected by me-
tabolism of the parent compound nor plasma protein
binding [71]. The extent of brain exposure is most of-
ten expressed as the B/P ratio (brain concentration
(AUC) / plasma concentration (AUC)) and is deter-
mined by the compound of interest’s preferential bind-
ing between brain tissue and blood [70]. Most clini-
cally available CNS drugs have a B/P of >0.3, that is
the compound concentration in the brain is 30% of the
total compound concentration in the plasma [7].
The most commonly used methods to predict BBB
penetration rate in cellular models utilize either Caco-2
[72] or Madin-Darby canine kidney (MDCK) cells [73,
74]. Caco-2 cells are human intestinal cells whose
membranes demonstrate many of the same transporters
and tight junctions as the BBB. However, despite these
similarities Caco-2 cell permeability is not a reliable
predictor for in vivo BBB penetration [75-77]. The ap-
plicability of MDCK cells has also been questioned due
to their origin from canine kidney capillaries not hu-
man brain capillaries. A modified MDCK cell line
transfected with the human MDR1 gene (which en-
codes for P-gp) has however, been shown to be a good
predictor of CNS drug effects on 93 clinical CNS com-
pounds, including P-gp substrate assessment [78]. As a
result of this transfection MDR-MDCK cells are the
1398 Current Medicinal Chemistry, 2016, Vol. 23, No. 14
most often employed in vitro predictor of P-gp sub-
strate potential [79].
Much effort focuses on improving microvessel en-
dothelial cell permeability assays. These are based on
isolated brain microvessels from a variety of species
and the resulting culture of endothelial cells [80].
When cultured and plated they form monolayers with
tight junctions [81]. There are several drawbacks to this
method not least of which is that the cells are used as
primary cultures. This presents the need for a supply of
fresh brain, rapid preparation and use, and a specialized
skillset. More importantly the expression of transport-
ers varies with each preparation leading to substantial
reproducibility and predictability issues [82, 83]. Addi-
tionally the brain capillary endothelial cell monolayers
display a transendothelial electric resistance (TEER)
lower than that of the BBB resulting in a much ‘leak-
ier’ model system [84].
Human pluripotent stem cells (hPSCs) are an area of
much concentration in developing cellular models of
the BBB. The hPSCs can be specified to express many
properties of the BBB including tight junctions, trans-
porters, and efflux transporters [85]. For further infor-
mation on this emerging area of BBB modeling the
reader is directed to several excellent reviews on the
subject [86, 87].
A frequently utilized and high-throughput method
of BBB permeability rate prediction is the PAMPA-
BBB method. This method uses porcine brain lipid in
dodecane as a model blood-brain barrier [88]. PAMPA-
BBB successfully predicted the ability of 30 commer-
cial drugs to either permeate or not permeate the BBB
when effects from active uptake/efflux transporters
were discounted [30]. The method was also used to
show significant correlation to in situ brain perfusion of
tested compounds leading to the PAMPA-BBB method
being a reliable, accurate, and low cost model for sim-
ple BBB-penetration prediction [70]. However, the
PAMPA-BBB model is a passive transport model, that
is without efflux transporters, making this model of
limited use to predict BBB transport in vivo if used in
isolation [89].
Several other methods have, and are, being devel-
oped for BBB penetration prediction including immo-
bilized lipid HPLC columns [90] and immortalized mi-
crovessel endothelial cell lines such as TR-BBB [91].
For more detailed information concerning in vitro
methods of BBB permeability prediction the reader is
directed to recent reviews [92, 93].
Paul C. Trippier
IN VIVO MEASUREMENT OF BBB PENETRA-
TION
Intravenous techniques performed in animal models
represent the ‘gold standard’ of BBB penetration
measurement as they take into account full physiologi-
cal conditions [94]. Early intravenous techniques relied
upon the brain uptake index (BUI) method which used
radiolabelled drug injected into the animal with triti-
ated water [95]. The tritiated water is used as an inter-
nal standard to allow calculation of the amount of
compound in the brain. This method is now mostly ob-
solete with newer methods developed to address the
limitations of BUI, namely low sensitivity and short
measurement time [94].
The in situ perfusion method addresses the limita-
tions of BUI [96]. In this method the direct catheriza-
tion of the common carotid artery of an anesthetized
subject animal is performed. Blood flow is stopped and
the perfusate (drug compound, oxygen, etc.) substitutes
for fluid flow to the brain. The integrity of the BBB has
been shown to be maintained during this experiment
which can be performed on a timescale of several sec-
onds to several minutes. The brain is removed from the
animal and the compound concentration in the brain is
measured (it should be noted that this method only per-
fuses one half of the brain – the half that receives blood
flow from the common carotid artery). Perfusion times
can be extended up to one hour by addition of oxygen
carriers [97]. This technique has been shown to be
around twice as sensitive as the BUI method, attributed
to the enhanced exposure time of the compound to the
BBB. The method can be extended to assess the trans-
porter dependence of compounds, with the comparison
of wild-type animals to transgenic animals that lack a
certain transporter e.g. P-gp deficient mice [98]. The in
situ perfusion method provides high-quality data but
requires considerable investment in time, surgery tech-
nique, and animals.
The mouse brain uptake assay is a rapid and rela-
tively inexpensive in vivo measure of BBB permeabil-
ity and brain distribution [99]. This method involves
injection into the tail vain of two mice. A mouse is sac-
rificed at five minutes and 60 minutes and the brain
removed. At five minutes there is minimum nonspe-
cific binding to brain tissue allowing accurate analysis
of BBB permeability. At 60 minutes it can be deter-
mined if the compound is cleared from the brain at the
same rate as the plasma, i.e. rapid equilibration across
the BBB. The two data points provide insights into ex-
act mechanisms of BBB permeability.
Selecting Good ‘Drug-Like’ Properties to Optimize Small Molecule
Brain microdialysis involves the implantation of a
stereotactic probe directly into the brain [100, 101].
Once the probe is implanted, multiple data points can
be obtained over the course of many days in animals
that are freely moving. Moreover, the method has been
used in humans to sample the concentration of drugs at
various time points. The data provided by this method
can provide a ‘real time’ picture of BBB penetration.
However, the surgery is traumatic, time consuming and
the probe can actually disrupt the BBB, engendering
leakage. In addition, highly lipophilic compounds have
been noted to absorb onto the membrane within the
probe.
Cerebrospinal fluid (CSF) measurement as an in
vivo method to assess BBB penetration is non-ideal
[71, 92]. The blood-CSF barrier (BCSFB) which regu-
lates compound permeability into the CSF is only about
1/5000th of the surface area of the BBB; compounds
detected in the CSF may have entered by the BCSFB
rather than the BBB [102]. In addition, the CSF is re-
freshed every five hours providing rapid turnover. The
prevailing consensus is that drug distribution to the
brain and CSF must not be considered identical [103].
Radiolabelling of compounds to predict BBB pene-
tration originated from quantitative radiography meth-
ods [104]. Intravenous injection of the radiolabelled
compounds into an animal model and subsequent har-
vesting of the brain can provide BBB penetration data
by measuring radioactivity. The method is particularly
suited for situations when assessment of localization is
critical, i.e. in brain tumors [105]. Recent advances
have resulted in the development of radiolabeled P-
glycoprotein substrates as positron emission tomogra-
phy (PET) tracers allowing pharmacokinetic assess-
ment of P-gp substrate specificity [106, 107].
The use of animal-dedicated positron emission to-
mography (microPET) employing 11C radiolabelled
compounds of interest (which has the same chemical
properties as 14C but is detectable using PET) has es-
tablished itself as a superior technique for in vivo
evaluation of BBB penetration [108]. The technique
has been employed both in animals and human volun-
teers to trace the distribution of radiolabelled drug can-
didates [109]. An additional advantage of the PET
technique for neuroimaging is that it is not confined to
the 11C isotope. Fluorine is commonly encountered in
many drug molecules and may be replaced by the 18F
isotope which is a positron emitter and detectable by
PET [110, 111].
Use of radiolabelled compounds provides very de-
tailed and accurate information on distribution
Current Medicinal Chemistry, 2016, Vol. 23, No. 14 1399
throughout the brain. However, access to radionuclei
labeled analogues of compounds for these studies re-
quires specialized skillsets and relevant licenses.
It is obvious that in vivo methods to determine BBB
penetration are the most accurate, however the cost and
specialized skillsets required to use these methods pre-
clude their application to early stage drug discovery.
The in vivo models discussed above are all low
throughput and resource intensive, making them un-
suitable for early stage drug discovery where hundreds,
thousands, or tens of thousands of molecules are to be
screened. The use of an ex vivo insect brain may pro-
vide a viable alternative for higher throughput screen-
ing while retaining many of the advantages of an in
vivo model. An invertebrate ex vivo brain has been em-
ployed to screen approximately six molecules a day for
BBB permeability. Species differences seem to be
comparable, with the insect BBB containing the tight
junctions, lipophilic characteristics and transporters
encountered in the mammalian BBB [112]. Further de-
tails on all highlighted models and additional tech-
niques can be obtained from recent comprehensive re-
views [99, 113-115].
DATA ANALYSIS
No matter which method is used to assess BBB
penetration, data analysis is paramount. When compar-
ing sets of data for a library of drug candidates it is ob-
viously essential that all data is analyzed in the same
way using the same calculations to arrive at a defined
parameter. It is equally essential to ensure that com-
parative data for known CNS active drugs is available
and what parameter has been used to define ‘good’
BBB penetration. It is therefore especially important to
account for both compound penetration through the
BBB and active transport through (or out of) the BBB.
The ratio of brain to plasma concentration (Kp) is,
historically, the most widely used parameter to quantify
brain penetration, however its relevance has been ques-
tioned [71]. The range of values accepted as defining
‘good’ BBB penetration is particularly broad, with a
variability of up to 2000-fold [25]. In a study of the 32
most prescribed CNS drugs, most displayed a range of
Kp = 0.1-24 in mice, suggesting Kp alone is not an ef-
fective determinant of BBB penetration [7]. Protein
binding in both brain and plasma exerts an influence on
BBB penetration as was discussed above and a clear
understanding of this factor is required to assess BBB
penetration. Indeed, it has been shown that different
stereoisomers bind with different rates to plasma pro-
teins [116]. The two enantiomers of cetirizine (R and S)
1400 Current Medicinal Chemistry, 2016, Vol. 23, No. 14
show distinct values for Kp of 0.04 and 0.22 respec-
tively (Fig. 4). These values would suggest, at face
value, that S-cetirizine would penetrate the BBB better
than the R-isomer. However, when BBB permeability
was evaluated by using the unbound concentration ratio
(Kp,uu) both stereoisomers were shown to possess iden-
tical penetration.
Fig. (4). Chemical structures of Cetirizine. The R- and S-
isomers show distinct values for ratio of brain to plasma
concentration yet penetrate the BBB in equal ammounts.
To avoid such confusion and false-positives/ nega-
tives in the future, the drug discovery community is
now moving to a definition of extent of BBB penetra-
tion that is defined as the ratio of free brain concentra-
tion and free plasma concentration at equilibrium (Kp,
free or Kp, uu) [16, 23, 117].
Kp,uu can be derived at steady state from the three-
compartment model:
Where PS is the permeability surface area product;
CLin is the active uptake into the brain; CLout is the ac-
tive efflux out of the brain; CLbulk is clearance due to
brain interstitial fluid bulk flow; CLmetabolism is brain
metabolic clearance [113].
Relevant in vivo estimations of drug delivery to the
brain can be described by three parameters: a) CLin to
account for active transport into the brain, b) Kp,uu to
account for the ratio of unbound drug in the brain (and
thereby free to engage its target providing therapeutic
effect [118]) to that in the blood and c) Vu,brain to ac-
count for intra-brain distribution [25]. It is recom-
mended that a combination of these three parameters
are measured for each compound in the drug discovery
program, as a single method will fail to take into ac-
count all variables. Among known CNS drugs Kp,uu can
vary by up to 150-fold, and Kp can vary by up to 2000-
fold.
Paul C. Trippier
OUTLOOK
Unbound-Drug Brain Exposure Assessment
It is widely agreed upon that a concentration of un-
bound-drug brain exposure at, or exceeding, pharmaco-
logically relevant levels is a critical requirement for
NCEs to show therapeutic benefit in CNS disorders and
may very well be the most relevant measure of BBB
penetration [25]. The high-throughput equilibrium di-
alysis-based assay for estimating the fraction of un-
bound drug in the brain and measuring complete brain
concentration of a compound of interest, promised to
usher in a new era in predicting successful CNS drugs
[123]. However, this assay utilized brain homogenate
that by its very nature changes brain tissue-binding
properties that introduce errors into datasets from this
technique [124]. Estimates place the error within 3-fold
when used as a surrogate to predict brain concentration
[125]. As such, while a useful model, its accuracy has
fallen short of expectations.
Recently the brain slice method has been applied to
assess drug distribution in the CNS [126]. This has the
substantial benefit of retaining close to normal physio-
logical conditions in terms of pH gradients, cell-cell
interactions and transport systems, providing an in vitro
tissue model very similar to an in vivo brain. The
method allows a simple, high-throughput, and cost ef-
fective technique to determine unbound volume of dis-
tribution in the brain [127]. There is a great deal of an-
ticipation that this method which measures binding and
distribution of a compound within the brain, when
combined with other techniques to measure BBB
transport, will be highly successful in predicting BBB
penetration of NCEs [115].
The exact relationship between physicochemical
properties of drugs and unbound-drug CNS exposure
has yet to be defined [32]. A recent study of 43 struc-
turally diverse CNS drugs was analyzed for unbound-
drug concentration in the brain [32]. The study found
that hydrogen bonding unequivocally influenced un-
bound-drug concentration in the brain, while lipophilic-
ity showed no effects – the level of unbound brain ex-
posure is influenced by structure. Use of the brain slice
method to define structural characteristics that influ-
ence unbound-drug concentration in the brain is ex-
pected to result in new insights for prioritizing phys-
icochemical properties to ensure NCEs remain un-
bound.
In silico methods to predict unbound-drug concen-
tration in the brain are under development and are
becoming increasingly more reliable. A 89% prediction
Selecting Good ‘Drug-Like’ Properties to Optimize Small Molecule
rate with one model was observed on a test set of 111
marketed CNS drugs [128]. A computer model based
on lipid binding and pH partitioning simulations has
recently been described to predict brain exposure based
solely on chemical structure [129].
P-glycoprotein Substrate Prediction
Predictive methodologies for many aspects affecting
BBB penetration are at, or approaching an advanced
stage. The area that is being left behind however, is
arguably the most important – that of BBB influx and
efflux transporter substrate prediction. While predic-
tions of P-gp substrate specificity are improving, the
large binding site of P-gp, which allows multiple bind-
ing modes across a wide range of chemical space re-
mains a significant challenge [130]. In addition, several
studies have reported that the breast cancer resistance
protein (BCRP) is more highly expressed at the BBB
than P-gp [131, 132]. However, significant expression
level difference has been observed across species
[133]. This protein functions to remove exogenous
compounds from the BBB in the same way as P-gp,
thus its high expression level represents a new problem
for BBB penetration prediction. This is especially so
when it is considered that modeling systems for this
transporter are under developed compared with those
for P-gp [134].
The success encountered in QSAR predictions on
substrate specificity for two BBB transporters - the glu-
cose transporter (GLUT-1) and the large-neutral amino
acid transporter (LAT1), where in silico prediction
models give good reliability, points to the possibility
that P-gp, BCRP and other BBB transporters will
succumb to accurate modeling predictions [135, 136].
Other Developments
Cell penetrating peptides as drug delivery technolo-
gies have been known since 2000 and are a promising
area for enhancing BBB penetration of CNS therapeu-
tics [137]. The guanidinium group was determined to
be the active constituent for BBB and cellular mem-
brane permeation and a wide variety of guanidinylated
scaffolds have been utilized from peptides to carbohy-
drates [138]. These molecular transporters have been
used to deliver small molecules and proteins across
biochemical barriers. As a drug delivery method these
and other methods for penetrating the BBB are valu-
able assets for CNS therapeutic development however,
they currently suffer from a lack of generality in the
molecules they can transport. This will be an area of
continued and exciting progress in the near future with
Current Medicinal Chemistry, 2016, Vol. 23, No. 14 1401
the potential to provide a novel solution to penetrate
the BBB [139, 140].
Physical methods to temporarily open the BBB are
approaching clinical trial in humans. Microbubbles can
be made to vibrate by high-intensity focused ultrasound
that result in the endothelial cells that form the BBB
being pushed apart to allow the penetration of drugs.
This method has been successfully applied in murine
models with no adverse effects [141]. Should this tech-
nique prove successful and widely applicable in hu-
mans, the need to design small molecule clinical candi-
dates to fit specific physicochemical properties for
BBB penetration may become an artifact of the past.
It would seem that the field of CNS drug discovery
is at a pivotal moment when considering the definition
of good physicochemical properties for BBB penetra-
tion. Previous work by many individuals has focused
on optimizing penetration through optimization of
lipophilicity and removal of acidic functionality. How-
ever, this would appear to be only part of the equation
to ensure BBB penetration. The medicinal chemist has
developed many ways to penetrate the BBB, yet failure
in CNS drug discovery projects continues. This can in
no small part be attributed to efflux transporters at the
BBB removing substantial amounts of drug compound
from the brain, coupled with less than effective levels
of unbound drug concentration within the brain.
The ultimate aim of medicinal chemists is the de-
sign of compounds that can penetrate the BBB them-
selves in sufficient unbound brain concentration to
elicit a therapeutic effect.
OPTIMUM BBB PENETRATION – A FINE BAL-
ANCING ACT
There have been several attempts at defining ranges
of parameters to increase the probability of good BBB
penetration to several degrees of success. Lipinski’s
modified rules for BBB penetration are more accu-
rately guidelines and have served the medicinal chem-
ist well. However the traditional priority towards lipo-
philicty is now shifting more toward hydrogen bonding
as the major influence on a compound’s BBB penetra-
tion ability. The ratio of brain to plasma concentration
has been shown to have too broad of a range for pre-
dicting which compound will penetrate the BBB. The
ratio of unbound drug in the brain is widely accepted as
the best parameter for predicting BBB penetration [32].
However, the very basic attributes that control BBB
penetration are all related to the physicochemical prop-
erties of the NCE in question (Mw, logP, logD, PSA,
1402 Current Medicinal Chemistry, 2016, Vol. 23, No. 14
HBA, HBD). While these can be manipulated to en-
hance or reduce BBB penetration there is a self-
regulating upper limit of design. Almost all drugs in
clinical use are small molecules, while the exact mo-
lecular weight range for successfully penetrating the
BBB is debatable (<400, <500) it is undeniable that a
finite amount of space is available on a drug molecule
to optimize physicochemical properties. Within this
space three essential properties need to be tailored –
potency, solubility, and BBB penetration.
The intent in designing CNS active NCEs is that
they are effective therapeutics in treating the disease
they are designed to combat. To carry out this function
they must possess a pharmacophore – an exact spacial
arrangement of functionalities that display hydrogen
bonding suitable for binding the biological target of
action [119]. The pharmacophore represents an essen-
tial component of the molecule optimized over many
generations of compound analogues to be the most ef-
fective arrangement of functionalities. While it is hy-
drogen bonds that control BBB penetration they also
control potency and even a reduction of a single hydro-
gen bonding functional group can inactivate a com-
pound [120, 121]. There is therefore a minimum
amount of hydrogen bond acceptors and donors re-
quired for a drug compound to be active. This number
varies widely with target and compound structure.
In turn, solubility in aqueous media for delivery is
determined by hydrogen bonding and lipophilicity. A
compound’s ability to hydrogen bond to water allows it
to be soluble, a lack of hydrogen bonding interactions
leads to lipophilicity. A balance is required that does
not infringe on the pharmacophore if the drug is to
reach its target and be active. While different delivery
methods can counter high water solubility the patients
quality of life and compliance is ultimately the driving
force behind oral delivery.
It has been commented on that the majority of CNS
drugs contain amine functionalities. Simply adding
amines to a NCEs structure is not an effective strategy
to improve BBB penetration. Addition of an amine to a
phenyl ring will provide an aniline – a know toxi-
cophore that forms a variety of active species that bind
proteins [122]. Care also needs to be exercised in
avoiding intra-molecular hydrogen bonding that can
render a compound inactive [120].
It is therefore a careful balancing act between po-
tency, solubility, toxicity, and BBB penetration that is
required to design an effective CNS therapeutic. What
is the optimum BBB penetration? This is unique to
each individual molecule and can only be defined as
Paul C. Trippier
the optimized value for a particular compound. If the
drug in question is designed to act peripherally, the op-
timum BBB penetration would be zero as no undesir-
able CNS side effects would be possible if the com-
pound cannot penetrate the BBB.
After all the BBB penetration design parameters
have been applied and predictive models performed
there is one more aspect of BBB penetration that can-
not be ignored – serendipity – a compound may simply
‘get lucky’ and be a substrate for active transporters at
the BBB, or ‘unlucky’ if that transporter is P-gp.
CONCLUSION
Prediction of good drug like properties for BBB
penetration has evolved over the past decades to take
into account several distinct parameters – brain distri-
bution (including unbound-drug concentration in the
brain), permeability, and efflux transporters. It appears
that for each of these parameters, successful guidelines
for the development of a CNS drug have been devel-
oped with the exception of predicting BBB transporter
substrate specificity. These guidelines can be collated
into a priority table of physicochemical characteristics
that if followed will represent the highest chance of
successes in achieving BBB penetration. However,
caution is required so as to ensure that the pharma-
cophore of the drug candidate is not impeded. Success-
fully penetrating the BBB with an inactive compound
is just as bad as not penetrating the BBB with the most
active compound.
From the preceding review it is apparent that an up-
dated set of priorities can be constructed to enhance the
chances of maximum BBB penetration of NCEs di-
rected to the CNS:
1. Minimize the number of hydrogen bond donors (in-
creases rate of penetration and unbound-drug brain
concentration)
2. Exclude acid functionality (increases potential to
penetrate the BBB as a hydrogen bond donor and
ionizable group is removed)
3. Higher lipophilicity (increases rate of penetration,
no effect on unbound-drug brain concentration)
4. Small molecular weight molecules (<500 Da, in-
creases ability to penetrate the BBB)
The traditional guidelines of higher lipophilicity
equals better BBB penetration are called into question.
Certainly limiting hydrogen bond donors in the NCE
will impact both penetration and unbound-drug brain
concentration thereby enhancing overall BBB perme-
Selecting Good ‘Drug-Like’ Properties to Optimize Small Molecule Current Medicinal Chemistry, 2016, Vol. 23, No. 14 1403

ability. It is noteworthy that for a medicinal chemist the NCE = New chemical entity
question of higher lipophilicity or lower number of hy- P-gp = P-glycoprotein
drogen bond donors may be a matter of philosophy.
Lipophilic groups on molecules are lipophilic in nature PS = permeability surface area
due to their inability to form hydrogen bonds with wa- PSA = Polar surface area
ter. It is therefore possible that reducing the number of
QSAR = Quantitative structure-activity relation-
hydrogen bond donors or increasing lipophilicty result
ship
in the same net gain in BBB penetration. Other descrip-
tors for good drug like properties include PSA and TEER = Transendothelial electric resistance
logD, both of which are heavily influenced by the hy-
drogen bonding potential of the NCE in question. CONFLICT OF INTEREST
Perhaps the correct priority assignment is a matter The author confirms that this article content has no
of semantics and relies on the use of hydrogen bond conflict of interest.
acceptors – increases rate of BBB penetration, has
minimal effect on unbound-drug brain fraction and re- ACKNOWLEDGEMENTS
duces lipophilicity. A quick survey of the structures of We are grateful to Texas Tech University Health
clinically approved CNS drugs shows that most contain Sciences Center for funding.
several purely hydrogen bond acceptor functionalities
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