Current Medicinal Chemistry, 2006, 13, 1757-1775 1757

Delivering Drugs to the Central Nervous System: A Medicinal Chemistry or

a Pharmaceutical Technology Issue?
Maurizio Ricci* , Paolo Blasi, Stefano Giovagnoli and Carlo Rossi

Dipartimento di Chimica e Tecnologia del Farmaco, via del Liceo 1, 06123, Perugia Italy
Abstract: This review aims to summarize the non-invasive approaches employed in delivering drugs to the
central nervous system which is severely hindered by the presence of the blood–brain barrier (BBB) that limits
molecular permeation. Particular attention will be placed on the several available strategies for delivering drugs
into the brain, through circumvention of the BBB, in order to critically address the medicinal chemistry and the
pharmaceutical technology contributions.
Keywords: Blood-brain barrier, brain targeting, chemical delivery systems, CNS delivery, SLN, pro-drugs, liposomes,
polymeric nanoparticles.

INTRODUCTION fluid of the brain). The CSF, produced mainly by highly

An almost alarming number of statistics, nowadays,
indicates that there is a startling increase of brain disease
incidence. In fact, in Europe alone, about 35% of the total
burden of all diseases is represented by brain pathologies [1].
Moreover, it is estimated that the market is largely under-
penetrated by central nervous system (CNS) drugs and
therefore, just to become comparable to the global market for
cardiovascular drugs, the CNS drug market should grow by
about 500% or more [2, 3]. Delivering drugs to the CNS is
not an easy task and the presence of the BBB is one of the
main problems in CNS drug development [2].
vascularised ependymal structures, namely CP, fills the
ventricles and the subarachnoid space and is in direct
relationship with the interstitial fluid (ISF) of the brain. The
function of these barriers is to provide a better homeostasis
of the CNS than in peripheral tissues. In fact, the
concentration and clearance of molecules, both endogenous
and exogenous, essential for the normal function of the
brain, are strictly regulated by the anatomic and physiologic
features of the barriers [4].
1.1. Choroid Plexus

This review will focus on the several available strategies
for delivering drugs into the brain, through circumvention of
the BBB issues, in order to critically address the medicinal
chemistry and the pharmaceutical technology contributions.
An introduction, containing the fundamental biological
background concerning the BBB will be provided first,
followed by a critical report of the “medicinal chemistry-
based” and “pharmaceutical technology-based” approaches.
1. CENTRAL NERVOUS SYSTEM BIOLOGY
The CP is a leaf-like highly vascularized structure laying
in the ventricles. The surface area developed by this structure
has been incorrectly believed to be 1/1000 or less of the
surface area developed by the BBB [5]. For some decades,
this misunderstanding has caused the CP to be neglected as
a gateway for delivering active compounds into the CNS.
Drug transfer in the CP is a complex subject in and of itself,
and it is out of the aims of this review. This topic has been
recently treated in an excellent issue of the Advanced Drug
Delivery Review [5].

The CNS is composed of the encephalon or brain and the
medulla spinalis or spinal cord. The brain is protected and
encased by the skull bones, while the spinal cord is
protected and encased by vertebrae. The two portions are
continuous with one another at the level of the upper border
of the atlas vertebra. The brain is one of the most, if not the
most, protected organ in the human body. In fact, specific
interfaces with the systemic circulation tightly regulate the
exchanges between the peripheral circulation and the
cerebrospinal fluid (CSF) circulatory system. The
aforementioned interfaces are: i) the choroid plexus (CP)
epithelium (blood-ventricular CSF); ii) the aracnoid
epithelium (blood-subaracnoid CSF); and, iii) the BBB
formed by the cerebrovascular endothelium (blood-interstitial
*Address correspondence to this author at the Maurizio Ricci, via del
Liceo 1, 06123, Perugia Italy; Tel: +39 075 5855125; Fax: +39 075
5855163; E-mail: riky@unipg.it
1.2. Blood-Brain Barrier
BBB is for sure the most important barrier involved in
the regulation of molecules accessing the brain. In fact,
while the volume occupied by capillary and endothelial cells
is about 1% and 0.1% of the brain volume respectively, the
brain microvasculature develops a total surface area of ~ 20
m2 (~ 100 cm 2/g of tissue) and a total length of ~ 400 miles
[6]. This high vascularization confers to the brain a very
intricate network of capillaries with an average distance of ~
40 µm [6]. This means that every single brain cell will be
placed virtually at no more than 20 µm from a capillary,
thus creating a space that can be permeated (simple
diffusion) by small molecules in a half a second [6].
Therefore, once it has crossed the BBB, a molecule can
virtually be distributed to all areas in the brain.
Unlike peripheral capillaries, those of the brain present
no fenestrae and the brain capillary endothelial cells

0929-8673/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd.

1758 Current Medicinal Chemistry, 2006, Vol. 13, No. 15
(BCECs) are sealed together by particular tight junctions
(TJs) or zonula occludens, and have a low amount of
pinocytosis vesicles [7-9]. TJs are structures that form a
narrow and continuous seal that surrounds each endothelial
and epithelial cell at the apical border aimed at strictly
regulating the movement of molecules through the
paracellular pathway [10, 11]. Even if TJs are quite similar
in all barrier systems, BBB endothelia TJs show some
differences in morphology, composition, and complexity
features from those of both the epithelia and peripheral
endothelia [12, 13]. TJs are made up of three integral
membrane proteins: occludin, claudin, and junction adhesion
molecules as well as different cytoplasmic accessory proteins
and lipids [9, 11, 12]. However, information about how
proteins and lipids are interconnected together within the TJs
structure and how this will affect the barrier properties is
still lacking [12, 13]. These structures, together with the
BCECs, form an almost impenetrable (or impermeable)
barrier for drugs administered by the peripheral circulation.
A contribution to the barrier function of the BBB is also
given by the periendothelial accessory structures represented
by the pericytes (PCs), the astrocytes (ACs) and the basal
membrane Fig. (1). The basal membrane supports the
endothelial cells in one side and the astrocytes and the
pericytes in the other side Fig. (1). ACs are the most
abundant cell type in the brain and, traditionally, they are
classified into fibrous and protoplasmic ACs on the bases of
their morphology and localization. Fibrous ACs have a star-
like morphology and often present many long processes,
also known as “end-feet” that end on the basal membrane of
the cerebrovasculature Fig. (1). ACs have been found to
Fig. (1). A representative cross-section of a cerebral capillary of the BBB (adapted from: Expert Reviews in Molecular Medicine ©
2003 Cambridge University Press, http://www.expertreviews.org/).
Ricci et al.
contribute to the brain homeostasis in many ways. One of
the main functions is to buffer the extracellular environment
to maintain the K+ levels [14]. Moreover, ACs remove and
inactivate a variety of neurotransmitters from the
extracellular space [14], regulate and produce growth factors
and a number of cytokines implicated in the immune and
inflammatory response in the CNS [15]. ACs also provide
the brain with ApoE [14]. More important for the aims of
this review, however, is the fact that many in vitro [16, 17]
and in vivo [18] experiments have shown that ACs seem to
be necessary to elicit the peculiar characteristics of the BBB
[19, 20]. It seems that both the direct contact between
BCECs and ACs, and the production and release of soluble
factors by ACs are involved in the regulation of BBB
competences [16-18]. To date, a number of in vitro BBB
and membrane transport models are available to aid in the
selection of compounds [21, 22]. Recently, it has been
pointed out that bovine brain endothelial cells (BBECs)
have a higher reliability than Mardin-Darby canine kidney
(MDCK) cells [23]. BBECs are being extensively used as a
model for in vitro BBB passive permeability and membrane
transport studies due to their size, availability and the ease
with which pericyte free clones can be obtained and
subcultured.
PCs are spherical cells that contain a prominent nucleus,
many primary and secondary lysosomes in the cytoplasm [8,
24] and are very important constituents of the BBB. It
seems that these cells are virtually involved in all the
processes of BBB formation, differentiation, and integrity
[8, 24]. Unfortunately, their role is still not clearly and
completely understood because a specific marker for this cell
Delivering Drugs to the CNS
type is still lacking. PCs have been implicated in all the
three stages of angiogenesis, in the formation of the basal
membrane and in the regulation of the paracellular
permeability. Finally, their well documented phagocytic
capacity could be interpreted as an emergency barrier in the
case of BBB rupture [8, 24]. In fact, PC lisosomal density
was seen to increase by the BBB hyperosmotic opening
[25].
Some of the aforementioned characteristics, because of
the insurgence of a disease, may be changed and drug BBB
permeabilities might also be modified [26]. It is not an aim
of this review to discuss the special pathological conditions
of the brain that affect BBB characteristics and, thus, the
delivery of drugs under these circumstances that have been
recently reviewed [26, 27].
2. DELIVERING DRUGS TO THE CNS
The specific anatomic and physiologic characteristics
make drug delivery into the CNS a critical issue and, at the
same time, a very interesting and stimulating area of
research. Three main ways for delivering active molecules
into the brain have been proposed: intracerebral delivery,
intraventricular delivery and intravascular delivery.
The intracerebral delivery consists in bypassing the BBB
introducing thus the active molecules directly into the brain
parenchyma by drilling the bones of the skull. Even without
considering the invasiveness of the strategy, which is
obviously high, the characteristics of the brain parenchyma
make this approach unsuccessful. The tortuosity and the
tight packing of brain cells confer a very low diffusion
coefficient to both small and large molecules. For a protein
of about 20 kDa the diffusion coefficient is estimated to be ~
10-6 cm2/s [6]. Nerve growth factor, released from a
polymeric disk implanted in the brain, was able to diffuse
throughout the surrounding tissue only ~ 2-3 mm from the
surface of the polymer in one week [28]. Similar results have
been obtained with other drug delivery systems and actives
[29, 30]. However, due to the high invasiveness, this
approach may be taken into account in pathologies such as
cancer if the benefit/risk balance is sufficiently high [31, 32].
The intraventricular delivery is another strategy to bypass
the BBB. Since the CSF is in communication with the
interstitial fluid, the direct instillation of an active
compound into the cerebral ventricles will lead to a free
diffusion into the brain parenchyma. Unfortunately, in this
case the limited diffusion through the parenchyma is coupled
with a very fast and efficient CSF clearance, thus limiting
the usefulness of this strategy. Following the intraventricular
injection, a drug is generally more rapidly drained into the
systemic circulation than it is distributed to the interstitial
fluid [33]. Moreover, the dimension of the human brain for
such a low diffusion rate represents an additional limitation
to the feasibility of this approach.
Intravascular delivery remains, at least potentially, the
best solution. In fact, since each neuron is perfused with a
blood vessel, it can be hypothesized that this approach may
be able to deliver the drug to each single neuron everywhere
in the brain [34]. The main issue of the intravascular
approach is the presence of the BBB. Different approaches,
Current Medicinal Chemistry, 2006 Vol. 13, No. 15 1759
more or less invasive, have been developed to circumvent
this barrier so as to gain access to the brain from the
peripheral circulation.
Recently, the nasal route has been suggested as an
alternative strategy for bypassing the BBB, by shuttling
drugs directly into the brain through the olfactory nerve.
However, yet the usefulness of such a strategy is under
evaluation and a deep survey on this matter is far from the
aims of this review [35-38]*.
Some invasive strategies such as the osmotic or the
pharmacological opening of the BBB, will not be dealt
within this review [39, 40].
Usually, in non-brain capillaries, hydrophilic molecules
cross the endothelia by the paracellular or transcellular route.
As previously mentioned, the BBB anatomic characteristics
do not allow the diffusion of water soluble molecules via
these routes. Since it is known that some molecules present
in the blood, such as nutrients or exogenous compounds,
gain access to the brain interstitial fluid, other transport
pathways can be hypothesized. Histamine, a small
hydrophilic molecule of 111 Da, does not cross the BBB,
while small lipophylic or peculiar molecules, such as O2,
CO2, and ethanol, may freely diffuse through the BBB [41].
Lipid free diffusion through the BBB is one of the existing
ways of access to the brain, and it requires very strict
physico-chemical characteristics in order to be achieved,
namely: lipid solubility and a molecular mass lower than
400-500 Da [6]. Unfortunately, many drug candidates do not
present these intrinsic characteristics. It is estimated that
more than 98% of all potential neurontherapeutics cannot
cross the BBB to reach the proper brain molecular target [6].
Diazepam is an example of a drug that seems to reach the
brain by crossing the BBB through lipid free diffusion [42].
Another important issue is that most serious brain diseases,
such as Parkinson’s (PD), Alzheimer’s (AD), multiple
sclerosis and amyothropic lateral sclerosis do not respond to
small molecule therapeutics. Moreover, most drugs that
actually cross the BBB do not reach the CNS in
pharmacologically significant concentrations. Intermolecular
forces were also found to affect free diffusion through the
BBB [43]. In this regard, brain uptake has also been
correlated to lipophilicity, the presence of hydrogen bond
donors/acceptors, and rotatable bonds.
From a medicinal chemistry viewpoint, it is easy to
understand that BBB permeability becomes a critical issue
in the drug discovery process. One of the first attempts to
predict BBB permeability was made by Levin who
successfully correlated the octanol/water partition coefficient
to rat brain capillary permeability for drugs weighing less
than 400 Da [44]. The diffusion rate resulted, obviously,
inversely related to the molecular size and, of the 27
molecules studied, only vincristine and adriamicin escaped
this correlation [44]. It was reported that an optimal Log
octanol/water partition coefficient (Log P octanol/water) for
brain distribution and activity is in the range of 0-2 [45].
On the basis of these findings, the first envisaged
strategy to overcome BBB impermeability has been to
*Dal Piaz, A., Giunchedi, P., Bortolotti, F., Gavini, E., Colombo, G., Ferraro, L.,
Tanganelli, S., Scatturin, A., Menegatti, E., Colombo, P. AAPS Annual Meeting
and Exposition, Nashville (TN), November 6-10, 2005.
1760 Current Medicinal Chemistry, 2006, Vol. 13, No. 15
produce lipophilic analogues of active molecules without
compromising their activity. Unfortunately, this strategy is
not always applicable and, even though it allows rapid BBB
overcome, pharmacokinetic issues, such as plasma protein
binding enhancement and/or peripheral distribution, can
impair CNS uptake [46].
Greig et al. [47, 48] showed that out of seven
chlorambucil esters, intended as chlorambucil pro-drugs for
brain cancer treatment, only one accumulated into the brain,
while the other six showed increased plasma half-lives that
paralleled the increasing length and complexity of their ester
chain. It was speculated that this behavior could be related to
an increase of the long-chain ester binding to plasma
proteins [47, 48]. Moreover, recent studies have confuted the
validity of the octanol/water partition coefficient in
predicting BBB permeability [49, 50].
In silico (computer-based) approaches were also
employed to predict BBB permeability, as a tool to predict
either CNS bioavailability or toxicity [51-53]. Nowadays,
the use of in silico models to predict BBB permeability has
been limited, principally because of the restricted quality and
quantity of data (training set) used to build suitable models
[52]. Moreover, the presence of more than one way of
discriminating between drugs that can or cannot cross the
BBB (different transport mechanisms and not just lipid free
diffusion, see below), makes prediction difficult.
The BBB in the very beginning, in terms of drug
delivery, was imagined as a static lipid layer; recently, it has
been proved to be a dynamic interface between circulating
blood and brain that eventually regulates, in a well
programmed manner, exchange processes [6]. The discovery
of the presence of transporters (e.g. P-glycoprotein) in
different portions of the BBB surely represents the main
feature of this change of concept. In fact, the BBB presents
endogenous transport systems that are able to shuttle
nutrients into the brain and expel harmful molecules. The
BBB transport systems are generally divided into three
groups: carrier-mediated transport (CMT), active efflux
transport, and receptor mediated transport (RMT).
2.1. Carrier-Mediated Transport
The BBB has many transport systems at the surface of
the endothelial cells that are responsible for the nutrients and
endogenous compounds intake into the brain. Since some
drugs resemble endogenous compounds, and when they do
not they may be properly modified to mimic them, these
transport systems represent useful biological carriers to
shuttle drugs or pro-drugs across the BBB in therapeutic
concentrations.
Glucose has to cross the BBB in large amount since it is
the main source of energy for the brain [54]. Glucose cannot
freely diffuse (lipid free diffusion) through the BBB, due to
its unfavorable physico-chemical characteristics, therefore a
specific carrier must be present on the BBB endothelium. A
family of glucose transporter proteins (GLUTs) carries
glucose from the blood to the brain. Many GLUT isoforms
have been identified, characterized and grouped into three
classes [55]. Classes I and II include all the known brain
GLUTs. Among others, 45 and 55 kDa isoforms of GLUT-1
Ricci et al.
and GLUT-3 are believed to be the main glucose transporters
of the brain [54, 55]. GLUT-1, the first glucose transporter
identified in the brain, is expressed on both luminal and
abluminal faces of the BCECs [54, 56].
A drug-nutrient conjugate strategy has been proposed to
cross the BBB by using biological transport systems [57,
58]. Bonina et al. [58] were able to protect mice from N-
methyl-D-aspartate (NMDA)-induced seizures by
administering a glucose ester of 7-chlorokynurenic acid. 7-
chlorokynurenic acid, one of the most potent antagonists at
the glycine site of the NMDA receptor, has interesting
potential therapeutic activity as a neuroprotective agent in
the treatment of stroke and epilepsy [59]. However, because
of its physico-chemical characteristics, 7-chlorokynurenic
acid does not reach CNS therapeutic concentrations when
systemically administered [60]. It was speculated that a
facilitated transport through the BBB by GLUT-1 could be
responsible for the pro-drug accumulation in the brain. The
enzymatic cleavage that follows, releasing the parent drug,
could explain the antiepileptic effect [58].
Essential amino acid (AA) transport through the BBB is
due to the presence of specialized carriers as well. AA
transporters may be generally classified in terms of substrate
affinity into three groups: anionic, cationic, and neutral
specificity. A large neutral AA transporter, also named
System L, and a cationic AA transporter, called System y+ ,
are present at the BBB as Na + -independent transporters [61].
The L-type AA transporter 1 (LAT 1), one of the System
L transporters recently cloned [62], represents the major
route that cells use to uptake branched and aromatic AA
from the extracellular fluid [63]. LAT 1 is particularly
expressed in tissues, such as brain, spleen, and placenta;
while a weak expression has been found in colon and testis.
LAT 1 is also highly expressed in some tumor cell lines
[62]. Due to its broad selectivity, LAT 1 is thought to be
responsible for the transport of a number of drugs such as L-
dopa, α-methyl-dopa, melphalan, triiodothyronine, and
gabapentine [63]. L-dopa, a drug used in the treatment of
PD, is taken up into the cells by LAT 1 and it is
metabolized into the active compound dopamine. The drug-
nutrient conjugate strategy has also been successfully applied
to LAT 1. In fact, by conjugating L-tyrosine to the parent
drugs it was possible to overcome the BBB [57, 64]. The
antiepileptic drug gabapentine is an example of a molecule
that mimics a LAT 1 natural substrate. In fact, this drug is a
γ -AA and not a natural substrate of LAT 1 (e.g., α-AA) and
it is presumed that the aforementioned carrier transports the
molecule across the BBB [65].
A recently characterized Na+ -dependent vitamin C
transporter (SVCT) expressed at the CPs, namely SVCT 2
[66], was investigated as a possible gate to the brain [67,
68]. Dalpiaz et al. [68] showed that nipecotic acid
conjugates (ascorbic acid or 6-Br ascorbate), contrarily to the
parent drug, could protect mice from pentylenetetrazol-
induced seizures.
Other transporters, namely the monocarboxylic acid
transporters such as MCT 1 [69], and the nucleoside
transporter system, might also be suitable in designing
drugs that use this strategy to cross the BBB. Moreover,
even if the importance of the nucleoside transporter system
Delivering Drugs to the CNS
to the BBB for nucleoside permeability is still controversial
[61], the anticancer drug gemcitabine seems to be transported
by this carrier system [70].
2.2. Active Efflux Transport
The importance of the discovery of P-glycoprotein (P-gp)
on the surface of the BBB and its potential implication in
delivering drugs to the CNS may be envisaged from the
numerous studies present in the scientific literature [71-75].
P-gp is an ATP-dependent drug transport protein present at
the apical membranes of different epithelial cell types,
including those forming the BBB. P-gp are N-glycosylated
membrane proteins of ~1280 AAs with 12 theoretical
transmembrane segments. Two ATP binding sites are found
in the intracellular portion [72]. There are two isoforms of
human P-gp, that are referred to as MDR1 and MDR2 (also
called MDR3), and three isoforms in mice: Mdr1a (Mdr3),
Mdr1b (Mdr1) and Mdr2 [71]. The function of the P-gp is to
efflux drugs from the brain to the blood in order to avoid
their accumulation in the brain parenchyma. In fact, actives
(substrates of P-gp), that generally were not able to
accumulate in the brain, reached high brain concentrations
either in the case of a co-administration of a P-gp blocker or
in the case of P-gp deficient mice [72, 76].
P-gp were first investigated as transporters that could
confer multi-drug resistance (MDR) to cancer cells; in fact,
many anticancer drugs are substrates of P-gp (e.g.,
vincristine, anthracyclines, taxanes, etc.). Other drugs such
as cyclosporine A (CsA), digoxin, dexamethasone and
others, were also found to be transported by P-gp. It is not
clear how such different molecules could be recognized and
transported by the same protein; it seems that the only
common feature is the presence of hydrophilic and
hydrophobic moieties in different regions of the molecule
[72]. The modulation of P-gp, intended as protein function
or its gene expression suppression, has been proposed as a
strategy in cancer chemotherapy [77] and can be successfully
applied to solve the problems of low brain drug
accumulation or efflux (in this case it is wrong to speak
about BBB penetration or permeability) from within the
brain [72]. In this regard, systematic investigations of in
vitro permeability and P-gp-mediated efflux have been
attempted [78] in order to determine whether these factors
can distinguish between CNS and non-CNS drugs. Analyses
of a variety of calculated physicochemical properties revealed
that CNS drugs had fewer hydrogen bond donors, fewer
positive charges, greater lipophilicity, lower polar surface
area, and reduced flexibility compared to the non-CNS
drugs. However, these physicochemical properties also
appeared to occur in compounds prone to P-gp efflux [79].
Mahar Doan et al. [77] have shown that large sets of CNS
and non-CNS drugs overlapped in the passive permeability,
P-gp efflux, and physicochemical properties. However, P-gp
efflux proved to be a good discriminant for the selection of
possible CNS drug candidates. These results actually
revealed that a fixed rule for identifying a molecule as a
CNS or non-CNS drug is virtually non-existent and this is
largely due to the lack of knowledge concerning the
mechanisms involved in brain uptake. This is also the
reason for the many failures recorded in CNS drug
development and research programs.
Current Medicinal Chemistry, 2006 Vol. 13, No. 15 1761
2.3. Receptor Mediated Transport
RMT is a strategy that, as previously mentioned, uses
biologic BBB features to gain access to the brain. There are
some endogenous large molecules that are able to cross the
BBB by using a transport system mediated by certain
receptors present on the luminal side of the barrier. Insulin
(IR) and transferrin (TfR) receptors are highly expressed on
the brain capillary endothelium [6]. Many other receptors
such as insulin-like growth factor receptor (IGFR), leptin
receptor (LpR) and others may be used with the same aim
[78-85]. The corresponding ligands (peptides) could be used
as brain drug delivery vectors. The strategy consists in
binding drugs or drug containers to peptidomimetic
monoclonal antibodies (MAbs) to allow the receptor to
recognize and shuttle the construct into the brain like an
endogenous molecule [6].
3. GETTING ACROSS THE BBB: A MEDICINAL
CHEMISTRY CONTRIBUTION
Chemically modified drugs represent a completely new
therapeutic tool, in contrast with the traditional drug
discovery programs, that, over the years, have been pursuing
mainly the identification of more and more potent new drug
molecules. The advantages of this approach have been
widely reviewed over the years [86-88]. Naturally, the
techniques and the strategies involved in drug modification
have evolved along with a new understanding of the
mechanisms regulating brain uptake. At the present day,
chemically modified drugs can be divided into: a) pro-drugs
and polymeric pro-drugs, and b) chemical delivery systems
(CDSs). The latter group includes conjugated systems and
molecules having specific targeting attitudes as well as more
recent advanced gene delivery and antisense therapeutics.
3.1. Pro-Drugs and Polymers
Early approaches aimed at improving drug penetration
into the CNS were based on chemical modifications of
therapeutics in order to improve functional features to
overcome the BBB. These pro-drugs are capable of
exploiting BBB metabolism. This is the result of the
bioconversion of the pro-drug into the parent molecule by
several mechanisms posed to protect the CNS. Some of
these mechanisms, such as the esterase activation, adenosine
deamidase activation, phosphodiesters into phosphotriesters
conversion and oxidase activation have been described [22,
85]. As reported earlier, the pro-drug approach was
successful in delivering dopamine and γ -amino benzoic acid
(GABA) as tetrahydrobipyridine and tripivaloyl derivatives
respectively [89], while, less recently, L-3,4 -dihydroxy-
phenylalanine (L-DOPA) was coupled with a thiazolium
precursor, such as thiamine disulfide, and delivered to rat
brains [90].
Although as early as in the 1950s and 1960s drugs were
already linked to polymers to improve their efficiency [91,
92], only recently the classic concept of pro-drug has been
evolved towards that of polymeric pro-drug [87]. By linking
a drug to a polymer, a conjugate is obtained with a higher
hydrodynamic volume characterized by a slower renal
1762 Current Medicinal Chemistry, 2006, Vol. 13, No. 15
excretion, longer blood circulation and an endocytotic cell
uptake [93, 94]. To select polymers as candidates for drug
carriers, a number of requirements should be fulfilled: i)
availability of suitable functional groups for covalent
coupling with drugs; ii) biocompatibility (nontoxic,
nonimmunogenic); and iii) biodegradability or a molecular
weight below the renal excretion limit. The use of several
polymers has been reviewed [87]. Among the most
employed are the vinyl polymers such as N-(2-
hydroxypropyl)methacrylamide (HPMA) Fig. (2),
polypeptides and proteins, polysaccharides and polyethylene
glycol (PEG).
3.2. Chemical Modification for Receptor Mediated
Targeting to the CNS
Linkage of macromolecules to drugs proved to produce
an enhancing effect on their half-lives and targeting
properties by improving their penetration into cells.
However, in recent years, researches have moved on by
looking at systems that are capable of exploiting endogenous
transporters and mechanisms. The identification of new
potential targets on cell walls and endothelial tissues has
impacted outstandingly on brain delivery system design.
One approach that has been explored over the past two
decades involves the administration of endogenous ligands,
or their analogs, to increase BBB permeability by activating
the receptors located on the endothelial cells. Brain
endothelial cells possess numerous neurotransmitter and
peptide receptors on both luminal and abluminal membrane
surfaces. These receptors are coupled with traditional second
messenger systems involving calcium fluxes, G proteins and
various kinases and phospholipases and several endogenous
ligands, such as adenosine, arachidonic acid, leukotrienes,
histamine, and bradykinin that increase BBB permeability
CH3
C H2 C C H2
C O x
NH
C H2
CH OH
CH3
C CH
H2
Fig. (2). Structure of PHMPA copolymer containing adriamycin (PK1).
Ricci et al.
when administered systemically [95]. Unfortunately, the use
of endogenous ligands is generally compromised by both the
high concentrations of ligand required to increase BBB
permeability and the physiologic side effects which can be
severe enough to damage brain capillaries. The drawbacks
associated with the use of endogenous ligands have been
partly overcome by designing new brain-targeted CDSs.
CDSs represent a rational drug design approach that exploits
sequential metabolism not only to deliver but also to target
drugs to their site of action [96-98]. The CDS concept
evolved from the pro-drug concept in the early 1980s, but
was developed by further chemical modification with the
introduction of targetor moieties. These brain-targeted
systems, in addition to providing access by increasing the
lipophilicity, exploit the specific bidirectional properties of
the BBB to ‘lock’ inactive drug precursors in the brain on
their arrival. CDSs comprise a targetor (T) moiety that is
responsible for targeting, site-specificity and lock-in; and a
modifier function (F), such as a lipophilizer, that is
responsible for protecting certain functions by providing the
necessary molecular properties to prevent premature,
unwanted biotransformation. The CDS is designed to
undergo sequential metabolic conversions, by disengaging
the F moieties and finally the targetor, after fulfilling its site
targeting role. As an example, the 1,4-dihydrotrigonelline to
trigonelline conversion proved to be efficient and safe, as in
vivo oxidation occurs with direct hydride transfer and
without generating highly active or reactive radical
intermediates [99].
Furthermore, since the ‘lock-in’ mechanism works
against the concentration gradient, it provides more
prolonged effects. Consequently, CDSs can be used not only
to deliver compounds, that otherwise would have no access
to the brain, but also to retain lipophilic compounds within
the brain, as it has indeed been achieved, for example, with a
CH3
C
C O
NH
CH2
C O H3C C H3
CH
NH
CH2
C ONH CH CONH CH2 CO
HO NH
H3C O
OC H3 O OH O
OH
C
OH C H2 OH
O O
Delivering Drugs to the CNS
variety of steroid hormones. The system has been
investigated with a large variety of drug classes comprising
biogenic amines; steroid hormones such as testosterone,
dexamethasone and estradiol; antiinfective agents; antivirals
such as acyclovir, trifluorothymidine, ribavirin;
antiretrovirals such as zidovudine (AZT), 29,39-
dideoxythymidine, ganciclovir and cytosinyloxathiolane;
anticancer agents such as lomustine and chlorambucil;
neurotransmitters such as dopamine, and GABA; nerve
growth factor (NGF) inducers such as catechol derivatives;
anticonvulsants GABA, phenytoin, stiripentol, felodipine;
monoamine oxidase inhibitors such as tranylcypromine;
cholinesterase inhibitors; antioxidants; nonsteroidal anti-
inflammatory drugs such as indomethacin and naproxen;
anesthetics such as propofol; nucleosides such as adenosine;
and peptides such as tryptophan; leu-enkephalin analogue;
thyrotropin-releasing hormone (TRH) analogue; and
kyotorphin analogues Fig. (3) [96]. Targeted drug delivery
to the brain via phosphonate derivatives was also explored,
and the so-called anionic chemical delivery systems (aCDS)
were designed, synthesized, and evaluated for testosterone
and AZT [100, 101]. As reported earlier, an evolution of the
CDS approach is represented by the use of inhibitors that are
able to modify or suppress specific endogenous mechanisms
in order to favor brain uptake. As a result, stimulation of
bradykinin receptors has been obtained upon administering
the selective B2 bradykinin agonist Cereport (labradimil or
RMP-7) [102]. Although Cereport has a lower affinity with
the B2 receptor (relative to bradykinin), its potency is
equivalent to bradykinin in vitro and even more potent than
bradykinin in vivo [103]. The reason may reside in its longer
half-life (2–3 times) if compared to bradykinin. Among the
several barriers impeding drug entry into the CNS, P-gp in
the BBB represents one of the hardest to overcome. As a
result, many drugs are driven outwards in spite of their
Fig. (3). Schematic representation of the molecular packaging and sequential metabolism used for brain targeting of neuropeptides.
TRH-CDS is included to provide a concrete illustration for the targetor (T), spacer (S), peptide (P), adjuster (A), and lipophilic (L)
moieties. [From Ref. 96].
Current Medicinal Chemistry, 2006 Vol. 13, No. 15 1763
ability to cross the BBB. Many anticancer drugs follow this
fate. Consequently, the P-gp function was modulated by
using P-gp inhibitors such as CsA, valspodar and elacridar
which have been proved to significantly increase brain
concentrations of docetaxel in wild-type mice with respect to
those obtained in P-gp knockout mice [104]. The inhibition
of efflux mechanisms is not the only approach that has been
used to deliver drugs to the CNS. Several other transporters
have been identified and targeted to enhance BBB
permeability. Natural molecules, such as neuropeptides,
peptides that act on the brain or spinal cord, represent the
largest class of transmitter substances and have a
considerable therapeutic potential for many CNS diseases
[105]. Their delivery through the BBB is even more
challenging than the delivery of other drugs, because
peptides tend to be rapidly inactivated by peptidases. The
use of endogenous BBB transport systems can be considered
a key to solving peptide delivery issues. Conjugation of a
drug that is normally not transported through the BBB, to a
peptide or a MAb forms the so-called chimeric peptide.
Chimeric peptides undergo receptor-mediated transcytosis
through the BBB in vivo on one of the endogenous peptide
receptor systems localized within the brain capillary
endothelial plasma membranes. They can be regarded as
“molecular trojan horses” [6], which mask the carried drug to
the BBB environment, thus mediating its uptake into the
CNS.
The chimeric peptide technology has been used to deliver
both neurodiagnostic [106, 107] and neurotherapeutic
molecules [108, 109] across the rodent or primate BBB, and
could be used for BBB drug targeting in humans. In this
case the peptidomimetic MAb transport vector is genetically
engineered to remove the immunodominant murine
sequences that would produce immune reactions in humans
[110]. Parallel to this, analogues of leu-enkephalin [111],
1764 Current Medicinal Chemistry, 2006, Vol. 13, No. 15 Ricci et al.

thyrotropin-releasing hormone (TRH) [112-114] and
kyotorphin [115, 116] have been shown to be potential
agents for treating neurodegenerative disorders, such as AD.
Although there is an absence of clear evidence, an
association between hypothyroidism and AD is suggested.
Recently, it was found that subclinical hyperthyroidism
(reduced TSH level) in the elderly considerably increased the
risk of dementia and AD [117]. It has been concluded that
this is caused by the reduced levels of TRH in the brain.
Therefore, brain targeted delivery of TRH can be of potential
therapeutic value in AD. On such basis, the TRH analogue
pGlu–Leu–Pro–NH ([Leu2] TRH) has been used to improve
TRH delivery to the brain. In fact, this conjugate showed a
significant increase in the extracellular acetylcholine
concentration during its perfusion to the hippocampus in
rats, when delivered in much smaller quantities compared to
free TRH [118]. Compared to the unmodified peptide, the
conjugated system significantly improved the cumulative
effect of the treatment on extracellular acetylcholine levels in
rats.
As demonstrated by these promising results, drug
conjugation with endogenous or endogenous-like ligands
seems to represent the right direction in achieving successful
CNS drug therapies. However, such a goal cannot be reached
without the parallel development of a discovery program
aimed at identifying of new and efficient paths to the CNS.
Currently, many studies have increasingly been focused on
new receptors. In this regard, the peripheral benzodiazepine
receptors (PBRs) have been identified in various peripheral
tissues as well as in glial cells in the brain [119, 120]. They
are pharmacologically distinct from the central
benzodiazepine receptors (CBRs) which are associated with
GABAA receptors and mediate classical sedative, anxiolytic,
and anticonvulsant properties of benzodiazepines.
PBRs have been targeted by N-imidazopyridinacetyl-
melphalan conjugates and their corresponding ethyl esters in
melphalan-sensitive human (SF126, SF188) and rat (RG-2)
glioma cell lines [121]. These conjugates exhibited PBR
binding affinity with IC50 values ranging from 57 to 2614
nM. Using a computational approach it can be predicted that
these conjugates possess significant brain penetration.
Conjugate half-lives exceeded 28 h (phosphate buffer 0.05M,
pH 7.4). Conversely, under the same conditions, the
corresponding acids were found to undergo a fast cleavage

OH OH
N N
OH
N N
O O-
(a)
OH O OH O

OH
N
N
O
(b)
O
O C CH2 O P EG3400
O 2

OH
N
N
O
(c)
O H
O C CH2 N C O CH2 O PEG3400
O 2

OH
N
N
O
(d)
O
O C C H2 N(CH 3) C O C H2 O PEG3400
O
2

Fig. (4). Camptothecin and camptothecin conjugates. (A) Chemical structure of camptothecin lactone and carboxylate forms. (B)
Direct ester bond: CM-PEG-CPT. (C) Ester bond through a glycine residue: CM-PEG-Gly-CPT. (D) Ester bond through a sarcosine
residue: CM-PEG-Sar-CPT.
Delivering Drugs to the CNS
within a few minutes. Another example of successful
conjugation concerned the basic fibroblast growth factor
(bFGF) that, after monobiotinylation and coupling with
OX26 MAb, through streptavidin (SA), reached the rat TfR
[122]. The bFGF retained receptor binding affinity and
increased brain uptake following OX26-SA conjugation. The
bio-bFGF/OX26-SA conjugate protected cortical cell
cultures against hypoxia/reoxygenation insult in a dose-
dependent manner in vitro. Recently, camptothecin (CPT)
has been conjugated with PEG Fig. (4) having a molecular
weight of 3400 Da [123]. These new conjugates are very
water-soluble and hydrolyze at a pH-dependent rate to release
the active parent drug. An improved uptake of these
conjugates by cells in vitro, if compared to implants and
cytotoxicity toward gliosarcoma cells, was observed.
Furthermore, the expression level of the TfR on BCECs and
the endocytosis of 125Itransferrin (125I-Tf) by this receptor
was investigated [124]. The TfR was expressed on BCECs
and actively endocytosed Tf, making it a suitable target for
drug delivery to the BBB and the CNS.
To date, there is considerable evidence of new potential
targets for delivering drugs to the CNS. An example is the
TNF-related weak inducer of apoptosis (TWEAK) which is a
TNF family member mediating proinflammatory effects by
its receptor fibroblast growth factor-inducible-14 (Fn14). The
role of TWEAK/Fn14 in experimental autoimmune
encephalomyelitis (EAE) has been studied by protein
vaccination with TWEAK and Fn14 and recombinant
TWEAK-DNA, respectively. It was seen that blocking Fn14
signaling may represent a novel approach for the treatment of
CNS autoimmunity [125]. Moreover, other potentially good
targets for improving CNS delivery are the GABA A receptor
subtypes (α1-, α2-, α3- or α5-subunits in combination
with β- and the γ 2-subunit) [126]. In fact, these receptors are
benzodiazepine targets whose sedative and anxiolytic effects
have been separated on the basis of distinct GABAA receptor
subtypes. Furthermore, receptors that are exclusively
extrasynaptic, such as the α5GABAA receptors (memory and
learning) and the δ-GABAA receptor (high sensitivity to
ethanol and neuroactive steroids), may represent good targets
to elicit a new pharmacological response, as shown by the
anesthetic action of propofol mediated by β3GABAA
receptors [127]. Less recently, three genes encoding for c-Jun
N-terminal kinase (JNK), a member of the family of serine
and threonine mitogenactivated protein kinases (MAPK),
were identified in humans (JNK1, JNK2 and JNK3) and ten
isoforms resulting from the alternative splicing of these
genes were characterized. JNK3 is primarily localized in
CNS neurons, making it an attractive CNS drug target, as it
is involved in key mechanisms affecting neurological
diseases including PD, AD and stroke. The inhibition of
JNK3 is a potential route for treating these diseases. Finally,
low-density lipoprotein (LDL) receptors represent a group of
cell surface receptors that transport a number of
macromolecules into cells through a process called receptor-
mediated endocytosis. There are nine members in the
receptor family, which includes the LDL receptor, the low-
density lipoprotein-related protein (LRP), megalin, and the
very low-density lipoprotein (VLDL) receptor. A number of
studies have suggested that the LDL receptors are involved
in endocytosis of drugs and drug formulations including
aminoglycosides, anionic liposomes and CsA [128].
Current Medicinal Chemistry, 2006 Vol. 13, No. 15 1765
3.3. The Emerging Scenario
An enormous number of genes of unknown function
contained in the CNS is involved in inherited diseases.
Genomic studies and gene therapy are effective tools for
targeting such genes. Manipulation of gene expression has
progressed in the last few years. Already in the 70s,
Zamecnik and Stephenson [129] used antisense DNA to
target a specific messenger RNA (mRNA) and prevent its
translation into a protein, whereas recently the elucidation of
the RNA interference (RNAi) pathway has further improved
gene-silencing procedures. The development of antisense
therapeutics brought forth by fast and common strategies
which consisted in the use of bare synthetic antisense
oligonucleotides (AOs), have been overtaken by the
conjugation approach. As an example, AOs have been used
to inhibit G-protein expression. AOs have been representing
outstanding tools for the discovery of protein functions in
vitro and in vivo. In particular, direct administration of AOs
into the CNS has become a popular tool in the study of G-
protein-coupled receptors (GPCRs). AOs target nucleic acids
in a sequence-specific manner, to inhibit the expression of
the corresponding receptor protein. As a consequence, GPCR
function can be modulated in order to favor drug entry into
the CNS. Although many technical limitations have come
forward, the antisense approach possesses several advantages
over the conventional pharmacotherapy and transgenic or
knockout methods. The classical pharmacological receptor
approach inhibits the function of biologically active
proteins, whereas the aim of AOs is to inhibit the synthesis
of these proteins. However, the many drawbacks associated
with the use of bare AOs limit their therapeutic use [130]. In
this context, although doubts are emerging on the real
effectiveness and future hopes of the next AOs generation,
conjugation may partially aid in solving the issues related to
gene therapy [131]. At the present day, modified ribozymes
(RZs), DNAzimes (DZs) and RNA interferences (RNAi)
have successfully been applied to reduce gene expression in a
growing number of genetic disorders both for functional and
gene therapy studies. The success of such approach is strictly
dependent on the level of knowledge of the endogenous
mechanisms and molecules involved with. Unfortunately,
although many genes have been identified, their biological
role remains poorly characterized. Nowadays, application of
DZs, RZs and short interfering RNA as chemically
synthesized compounds, expressed from plasmids or viral
vectors, has been investigated and reviewed [132]. A number
of patents have been issued on the use of new systems for
nucleic acid and peptide delivery to the CNS [133-141].
Generally, viral vectors are the most efficient gene
transfer method to CNS tissue, but synthetic compounds are
also highly effective when combined with the right
transfection method. Plasmids have not been as successful
since they have given poor transfection efficiencies in
neuronal tissue probably because of their large size. Viral
vectors have extremely high transfection efficiencies;
however they elicit also an immune response in the brain
that can lead to the destruction of the transgene [142].
Chemically synthesized compounds are less immunogenic,
but the transfection efficiency is scarce. They are unstable,
their long-term expression is impaired, and they cannot cross
the BBB. For this reason, researches are now focused on
1766 Current Medicinal Chemistry, 2006, Vol. 13, No. 15
synthesizing stable compounds engineered to pass the BBB,
as well as the development of less immunogenic viral
vectors. In some cases, stabilization has been achieved by
the addition of phosphorothioate linkage, 2'-O-Me
nucleotides, 3'-terminal nucleotide inversions and locked
nucleic acids [143-145]. With the same approach, a
chemically modified RZs, Angiozyme TM, was targeted
against the vascular endothelial growth factor (VEGF)
receptor [146]. This system is currently in clinical trials at
Sirna Therapeutics (Boulder, CO, USA). Of course, a lot has
to be done to design and fabricate RZs that ensure BBB
crossing and effective transfection and long-term expression.
In this regard, different ongoing approaches are now being
investigated. Several new vectors have been designed, such
as adeno-associated viruses (AAVs), which are emerging as
promising delivery vehicles with low immunogenicity and
toxicity, while retaining long-lasting expression levels.
AAVs have been used to deliver therapeutic ribozymes to the
retina [147] and adenovirus short interfering RNA to the
brain in vivo [148]. A further interesting virus-based system
is a recently reported one that describes the use in vivo of
lentivirus in cycling and non-cycling mammalian and stem
cells [149]. Jin et al. [150] injected recombinant adenoviral
vectors expressing GFP intravenously into mice and these
vectors successfully crossed the BBB and reached the CNS.
GFP was expressed after 6 days, with a maximum at 14
days which diminished after 28 days. The GFP expression
was localized mainly in the hippocampal regions, but also
observed in the midbrain and cerebellum. This finding is
encouraging; however, the strong immune response
encountered after intravenous application and the clearing of
virus in the liver remain a major obstacle. Overcoming such
unwanted side effects is the next step, and the
implementation of new strategies, other than just chemical
modifications, is a forced and inevitable approach.
4. GETTING ACROSS THE BBB: A PHARMACEU-
TICAL TECHNOLOGY CONTRIBUTION
4.1. Colloidal Drug Carriers
While multiple strategies have been employed to
circumvent the BBB, an emerging, attractive, approach is
the use of nanosystems that are properly tailored to deliver
drugs to the brain [151-154]. In this respect, liposomes and
polymeric nanoparticles (NP) represent the most exploited
ones, although micelles, emulsions, solid lipid nanoparticles
(SLN) and nanostructured lipid carriers (NLC) have also
been studied [155]. The reason for using colloidal carriers is
to increase bioavailability to the brain by promoting their
diffusion through biological membranes. In fact, drug
entrapment within carrier systems protects the molecules
being delivered against the many mechanisms involved in
xenobiotic inactivation and clearance. Nevertheless, the
possible therapeutic benefits depend on their distribution
within the organism. After intravenous administration, all
colloidal systems cannot extravasate except in tissues with a
discontinuous capillary endothelium, such as the liver,
spleen, and bone marrow. Even in these organs, only
particles smaller than about 100 nm can penetrate into the
tissue, since this value is the approximate gap size between
endothelial cells. Conversely, colloidal carriers can
Ricci et al.
extravasate into solid tumors, as well as inflammed or
infected tissues where the endothelium is defective. After
intravenous administration, they interact with specialized
plasma proteins (opsonins), especially with immuno-
globulins (IgG), albumin, the elements of the complement,
fibronectin, etc. (opsonization process) and for this reason
they are rapidly (usually within minutes) cleared from the
blood stream [156]. The type and the extent of this process
mainly depend on particle size, charge and surface properties
[157, 158]. In order to avoid opsonization, some “steric
hindrance” strategies can be applied: by adsorbing at the
colloid surfaces some surfactant molecules, such as
copolymers of polyoxyethylene (POE) and polyoxypro-
pylene (POP), or by providing a sterical stability by direct
chemical linking of PEG to the particle surface [159, 160].
PEG coating is proved to prevent recognition of liposomes
by macrophages due to the reduced binding with plasma
proteins [161]. Unfortunately, a long circulating time does
not ensure an effective BBB overcome and, therefore,
additional carrier features are needed in order to deliver drugs
in therapeutic concentrations to the CNS. In this respect,
exploitation of specific CMT systems on the BBB can be
considered as a major strategy for delivering drugs as well as
drug loaded colloidal carriers, to the CNS. Among these
systems, the LDL-receptor and the transferring transcytosis
systems may be employed in the delivery of drugs [162].
4.2. Liposomes
Liposomes consist of one or more phospholipid bilayers
enclosing an aqueous space. Water soluble drugs can be
included within the aqueous compartments while lipophilic
and amphiphilic molecules can be associated with lipid
bilayers.
Liposomes represent promising drug carrier systems that
can be applied to human use because of their ability to alter
the pharmacokinetics of drugs which results in a reduction of
their toxicity. In this respect, liposomes have been
considered for site specific targeting in several brain
pathologies through both intracerebral and intravenous
administrations, although most of the studies have been
focused on tumor therapies. Nevertheless, after intravenous
administration, the pharmacokinetics of conventional
liposomes is characterized by a very high systemic plasma
clearance, since such vesicles are rapidly removed from the
circulation by macrophages of RES [163]. In general, this
high plasma clearance, strongly limits their usefulness as
site specific drug delivery systems. Liposome half-life can
be prolonged considerably, up to 90 h in humans [164], by
PEGylation (sterically stabilized liposomes) [165-168]. PEG
phospholipids are safe and can be prepared synthetically with
high purity and in large scale, thus allowing their use in
clinical applications. Although PEGylated liposomes (PL)
show a 200-fold decrease in systemic plasma clearance, from
22 to 0.1 l/hour, they are characterized, similarly to
conventional liposomes, by a low affinity to cells in vitro
and healthy tissues in vivo, especially brain tissue as they are
not transported through the brain capillary endothelial wall.
On the contrary, their long circulating time allows them to
selectively extravasate in pathological sites, such as tumors
or inflamed regions with a leaky endothelium, thus
improving the therapeutic index of encapsulated drugs [169].
Delivering Drugs to the CNS
Currently, several doxorubicin HCl containing PL
preparations are commercially available (Caelyx® [170],
Doxil®[171]) for clinical practice with some success against
glioblastomas and metastatic tumors with long-term survival
[172, 173]. Moreover, prednisolone loaded PL have been
effective against encephalitis [174]. Restoration of the BBB
integrity and diminution of the macrophage infiltration were
observed in the animal models. PL resulted highly effective
in the treatment of experimental autoimmune encephalo-
myelitis, and were superior to a 5-fold higher dose of free
methylprednisolone. A further improvement of PL brain
distribution can be obtained by conjugating an appropriate
targeting moiety that will be recognized by cell surface
receptors in the targeted tissue [175]. This “active targeting”
can be achieved by using modified proteins [176] or certain
receptor specific MAbs which undergo receptor-mediated
transcytosis through the BBB. In this regard, the OX26
MAb to the TfR or MAb to the IR has been used for this
purpose [177, 178].
Antibody-directed liposomes, namely immunolipo-
somes, can be prepared through two different coupling
strategies: i) covalent coupling of MAbs directly to the
liposome surface, ii) coupling to the distal tip of PEG
chains. Direct coupling of proteins to the lipid headgroup on
the PL surface results in a strong PEG chain steric hindrance
effect that prevents the antibody–target interaction [179]. In
this respect, several studies have proved the PEG chain
length importance in reducing immunoliposome target
binding effectiveness in vivo, of up to 50% [180, 181]. The
effect of PEGylation is less pronounced with PEG molecular
masses of 2000 or 750 Da, while reaches its maximum with
PEG of 5000 Da molecular mass. A PEG chain shielding
effect has also been demonstrated to increase with PEG
molecular weight through a comparison of antibody
dimensions (ranging from 10 to 15 nm) with the PEG
corona thickness, which for PEG2000 is about 5 nm in its
coiled state, and 15 nm when in a stretched form [179]. In
the light of these findings, a cell-specific ligand was bound
directly to the distal end of lipid-conjugated PEG molecules
rather than to lipid headgroups on the PL surface [182].
Thus, PEG is used as a spacer and this turns out in better
accessibility and flexibility to the ligand [183]. By using
this strategy, the immunoliposome target binding efficiency
both in vitro and in vivo increased by a factor of two to three
[184]. Different techniques have been developed for the
covalent binding of proteins to PEGylated phospholipids
[185, 186].
Huwyler et al. [162] proposed immunoliposomes to
deliver the antineoplastic agent daunomycin to the rat brain.
PEG-conjugated immunoliposomes (PIL) were prepared by
using thiolated MAb and a bifunctional PEG2000
containing distearoylphosphatidylethanolamine (DSPE) at
one end and a maleimide at the other end Fig. (5). The MAb
used was the OX26 MAb that is able to cross the BBB via a
mechanism involving the circulating transferrin transcytosis
receptor [187], which is abundant in brain microvascular
endothelium. Pharmacokinetics and brain uptake of
[3H]daunomycin was examined after i.v administration of
the drug as a free form, as conventional liposomes, as PL,
and as PIL. No brain uptake of PL carrying [3H]daunomycin
was observed demonstrating that the use of only sterically
stabilized liposomes confers no advantage in brain drug
Current Medicinal Chemistry, 2006 Vol. 13, No. 15 1767
delivery despite the marked increase in plasma AUC.
Conversely, the coupling of 30 OX26 antibodies per
liposome resulted in an optimal brain uptake. Saturation of
the delivery process was observed at higher anti-body
densities. The determination of brain levels of
immunoliposomes over 24 h revealed that immunolipo-
somes accumulate in brain tissue. Although brain uptake by
using immunoliposomes was 3- to 4-fold lower than the
values that can be obtained by using biotinylated drugs
directly attached to OX26-avidin analogue coniugates,
immunoliposomes increase the drug carrying capacity of
MAb [188, 189]. In fact, the OX26-avidin conjugate can
carry only 2-3 small molecules, whereas c.a. 28000
daunomycin molecules are entrapped into a single 100 nm
liposome, thus increasing the carrying capacity of the OX26
MAb up to 4 logarithmic orders of magnitude.
Fig. (5). Schematic diagram of coupling of the thiolated OX26
mAb with sterically stabilized liposomes containing DSPE-Peg-
maleimide (from ref. 187).
Immunoliposomes have also been used in brain tumor
gene therapy [190-192]. Most solid cancers, including brain
cancers, are dependent on the epidermal growth factor
receptor (EGFR) that plays an oncogenic role in 90% of
primary brain cancers such as glioblastoma multiforme
(GBM) and in about 70% of the peripheral solid cancers that
metastasize to the brain [193, 194]. Antisense gene therapy
directed at the gene EGFR is therefore a potential treatment
strategy. However, the development of brain cancer
therapeutics, which knock-down the function of the EGFR,
is strongly frustrated by the presence of the BBB perfusing
the primary or metastatic cancer. The main goal is thus to
find the right way to circumvent the multiple barriers
separating the bloodstream from the nucleus of the target
cell. Transvascular delivery of non-viral genes to the brain is
possible by using suitably tailored PIL [195]. Following
this approach, the plasmid, designated clone 882 which
encodes a 700 nucleotide RNA that is an antisense to the
nucleotides 2317–3006 of the human EGFR, was
encapsulated in PIL that were doubly targeted to brain
cancer, across biological barriers in vivo, with two MAbs of
different receptor specificities [196]. The first MAb was the
rat 8D3 MAb to the mouse TfR, which enabled the
triggering of the PIL receptor-mediated transcytosis across
the mouse BBB, but which does not mediate transport of
the PIL across the human brain cancer cell membrane. The
second MAb (mouse 83-14 MAb) targeted the human
insulin receptor (HIR) which enabled transport across the
1768 Current Medicinal Chemistry, 2006, Vol. 13, No. 15 Ricci et al.

plasma membrane and the nuclear membrane of the human
brain cancer cells without reacting with the mouse vascular
endothelial insulin receptor Fig. (6). Such a targeted gene
delivery system is characterized by a high stability of the
encapsulated DNA under physiological conditions, by a
prolonged duration of gene expression, and by an 88%
increase in survival time in adult mice, without toxicity after
chronic (weekly) i.v. administration [197].
This gene therapy strategy represents the only way to
deliver genes to the brain. In fact, cationic DNA polyplexes
(i.e., lipofection), in spite of the promising results obtained
in terms of the levels and duration of gene expression [198],
are still ineffective in delivering genes to the brain after in
vivo administration, mainly because they do not cross the
BBB [199]. Moreover, DNA-cationic liposomes form micro-
aggregates, particularly at lipid to DNA ratios where the
overall charge of the complex is neutral. As a consequence,
their pharmacokinetics and tissue distribution are
characterized by very short plasma half-lives (e.g., a few
minutes in the mouse) and an unspecific accumulation in
different organs. For these reasons, cationic liposomes
normally require an invasive administration [200]. Studies
on clinical gene therapies for malignant glioma by liposomal
delivery system are very few. In this respect, in April 2000,
Yoshida et al. [201] started the first clinical trials on safety
and effectiveness of IFN-β gene therapy in patients with
malignant GBM by using cationic liposomes [202]. This
study has shown the feasibility and safety of interferon β
gene therapy, which may become an important treatment
alternative for patients with malignant glioma. Another
strategy is based on the sensibilization of cancerous cells to
drugs. This approach involves liposomal delivery of the
herpes simplex virus thymidine kinase (HSV-tk) gene into
the glioma to improve cell sensitization to ganciclovir [203,
204].
The many studies regarding liposome utilization in brain
drug delivery have demonstrated their applicability in gene
therapy. However, some concerns are emerging about the
effectiveness of PL upon multiple successive admini-
strations. In fact, an extremely high clearance rate increase is
being observed upon second administration of stealth
liposomes. This has been ascribed to a preferential IgM
binding to PEG after the first dose [205, 206]. Hence, the
application of such systems has yet to be carefully
investigated in order to assess the main features that are
responsible for such a behavior.
4.3. Nanoparticles
The term nanoparticles is used for a well-defined drug
carrier system, generally (but not necessarily) of a polymeric
nature ranging in size between approximatively 10 and 1000
nm (1 µm) [207]. This definition encompasses either
nanocapsules, with a core-shell structure (a reservoir system),
or nanospheres (a matrix system). Since it is often difficult
to prove whether these particles have a continuous matrix or
a shell-like wall, the more general term NP is thus usually

Fig. (6). Schematic representation of a PEGylated immunoliposome (PIL) (A) PIL includes approximately 2000 strands of 2000 Da
PEG conjugated to the surface of the liposome to minimize rapid uptake by the RES. Approximately 2% of the PEG strands are
tethered at the tips of the strands with either the 8D3 or the 8314 MAb to cause binding of the PIL to either the mouse TfR or the
human IR, respectively. (B) Multiple barriers must be circumvented before a therapeutic gene injected in the bloodstream can
distribute to the nuclear compartment of the cancer cell. The 8D3 MAb to the TRFR causes receptor-mediated transcytosis of the PIL
across the tumor microvascular barrier, or BBB. The 83-14 MAb to the IR mediates transport of the PIL across the tumor cell plasma
membrane and the nuclear membrane of the tumor cell. (C) Electron microscopy of the PIL conjugated with the IR MAb, and
complexed with a conjugate of 10 nm gold and an anti-mouse secondary antibody. Magnification bar, 20 nm. (from ref. 196).
Delivering Drugs to the CNS
accepted. Drugs or biologically active materials may be
entrapped, encapsulated into NP and/or adsorbed or attached
on their surface.
NP show some advantages over other colloidal carriers
such as liposomes: i) their preparation methods are generally
simple and easy to scale-up [208]; ii) they are highly stable
both during storage and in vivo [209]; and iii) the
biodegradable materials used for their preparation allow
sustained drug release at the targeted site over a period of
weeks after injection.
Although NP can be made of a broad number of
materials such as poly(alkylcyanoacrylates) (PACAs),
poly(methylidene malonate), polyesters such as poly(lactic
acid) (PLA), poly(glycolic acid), poly(ε-caprolactone) and
their copolymers, polysaccharides, proteins, etc.,
poly(butylcyanoacrylate) (PBCA) proved to be, so far, the
most useful in preparing NP for in vivo delivery of drugs to
the brain, because of its rapid biodegradability [210].
However, polymeric NP show the same drawbacks of
liposomes for an effective drug brain delivery and several
attempts have been made to change their body distribution.
The most promising results were obtained by coating NP
with hydrophilic surfactants or by covalently linking POE
chains on their surface. In vivo studies performed by Kreuter
et al. [76, 152, 211, 212], unequivocally demonstrated that
a successful targeting to the brain of the hexapeptide
dalargin, a Leu-enkephalin analogue with opioid activity,
was obtained only by overcoating drug adsorbed PBCA NP
with different polysorbates, especially Tween® 80. It is
important to note that: i) a simple mixture of the drug with
NP, drug with Tween® 80 or a mixture of all three
components, mixed immediately before injection, were not
able to induce any effects on the CNS; ii) dalargin bound to
the NP also exhibited no effects; and iii) the transport of
dalargin also cannot be ascribed to a simple surfactant effect
on the endothelial cells or on the tight junctions, since most
of the surfactants investigated, such as poloxamers 184, 188,
338, 407, poloxamine 908, Cremophor EZ, Cremophor
RH40, and Brij 35, were inactive [211]. These findings
suggested that the overcoating of the NP with the
polysorbates was critical in leading a characteristic alteration
of the NP surface properties, responsible for specific
adsorption of certain plasma components and hence of the
uptake by the endothelial cells [213].
Studies on the pharmacokinetics of 3H-labeled dalargin
bound to PBCA-NP overcoated and unovercoated as well as
free 3H-labeled dalargin have shown the importance of NP in
enabling dalargin to cross the BBB [214]. In fact, the
plasma radioactivity was higher, up to 20 min with both NP
preparation and over 60 min with overcoated NP.
Other drugs have been transported across the BBB using
the polysorbate 80-coated PBCA NP such as kytorphin (a
dipeptide); loperamide [215]; tubocurarine [216]; MRZ
2/576 (NMDA-receptor antagonists) [217]; and doxorubicin.
MRZ 2/576 (8-chloro-4-hydroxy-1-oxo1, 2-dihydropyrid-
azino[4,5-b]quinoline-5-oxide choline salt) is a novel
NMDA receptor antagonist, characterized by a potent but
rather short anticonvulsant action (5–15 min) following
intravenous administration to mice as estimated by the
prevention of the maximal electroshock induced
convulsions. The MRZ 2/576 short activity is probably due
Current Medicinal Chemistry, 2006 Vol. 13, No. 15 1769
to its rapid elimination from the CNS by efflux pump-
mediated transport processes inhibited by probenecid.
Probenecid pretreatment prolonged the anticonvulsive
activity from about 15 to 150 min. Intravenous
administration of the drug bound to PBCA NP overcoated
with polysorbate 80, however, prolonged the duration of the
anticonvulsive activity in mice up to 210 min and after
probenecid pretreatment up to 270 min compared to the 150
min with probenecid and MRZ 2/576 alone. These findings
demonstrate that polysorbate 80-coated PBCA NP can be
useful not only in delivering drugs to the brain, but also to
prolong CNS drug availability.
Doxorubicin is one of the most important anticancer
drugs, but it is not used against brain tumors because it
cannot cross the BBB. Gulyaev et al. [218] observed that
doxorubicin bound to NP coated with polysorbate 80 was
found in a remarkable concentration (6 µg/g) in the rat brain
after intravenous injection at a doxorubicin level of 5 mg/kg.
Rats with intracranially transplanted glioblastomas 101/8,
treated with doxorubicin bound to polysorbate-coated NP
had significantly higher survival times compared with all
other groups. Over 20% of the animals in this group showed
a long-term remission [219]. Additionally, binding to NP
may reduce the systemic toxicity of doxorubicin.
To explain the mechanism involved in the transport of
drugs across the BBB by means of overcoated NP, different
hypotheses have been postulated [220]: 1) increased NP
retention in the brain blood capillaries combined with an
adsorption to the capillary walls, creating a higher
concentration gradient, and thus enhancing transport across
the endothelial cell layer; 2) a general surfactant effect
leading to membrane fluidification and an enhanced drug
permeability through the BBB [221]; 3) NP could lead to an
opening of the tight junctions between the endothelial cells
favoring drug penetration; 4) NP may be endocytosed by the
endothelial cells followed by drug release and diffusion into
the brain; 5) the NP with bound drugs could be transcytosed
through the endothelial cell layer; 6) the polysorbate 80,
used as coating agent, could inhibit the efflux system,
especially P-gp. However, Kreuter et al. [222] did not
support Olivier’s hypothesis that PBCA NP deliver drugs to
the brain by a nonspecific disruption of the BBB, either by
opening the tight junctions or by general toxic effects on the
BBB endothelium as a result of NP degradation [221].
Moreover, the fact that dalargin has to be pre-adsorbed onto
NP before it shows an antinociceptive effect suggests
specific delivery mechanisms to the CNS rather than a
simple physical disruption of the BBB. More recently, it
was demonstrated that the transport of loperamide-loaded
human serum albumin NP across the BBB, is mediated by
apolipoproteins (Apo) E (ApoE) and/or B (ApoB) adsorbed
to the NP after injection into the bloodstream or bound
covalently to their surface as well [223]†. This effect was not
obtained by coating NP with other surfactants besides
polysorbates. Coating of the dalargin-loaded NP with Apo E
or B without polysorbate 80 induced the antinociceptive
effect but at a lower level than when coated with polysorbate
80 alone. However, the antinociceptive effect was even
higher when the NPs were first coated with polysorbate 80
†Kreuter,
J.; Michaelis, K.; Dreis, S.; Langer, K. 15th Int. Symp. on
Microencapsulation, Parma (Italy) September 18-21, 2005.
1770 Current Medicinal Chemistry, 2006, Vol. 13, No. 15
and then overcoated with ApoE or B. It can be assumed that,
due to the interaction with ApoE, the NP would mimic
natural lipoprotein particles which could interact with the
LDL receptor family located in the BCECs followed by
endocytotic uptake. This was demonstrated by the fact that
in ApoE-deficient knock-out mice the antinociceptive effects
with dalargin-loaded polysorbate 80-coated NP were reduced
by about 50%. However, whether drugs are released within
the endothelial cells and reach the other brain compartments
by diffusion or whether exocytosis of NP with bound drug
occurs has not yet been well established.
Although PBCA NP are currently considered the most
suitable carriers, different engineered NP have been proposed
as brain targeting systems.
Olivier et al. [224] have synthesized OX26 conjugated
PEGylated NP using PLA with the aim of specific brain
targeting by TfR-mediated transcytosis. The same strategy
was used by Aktas et al. [225] who formulated PEG-
overcoated chitosan NP conjugated with OX26 using the
avidin-biotin approach, and evaluated their in vivo
effectiveness. Lu et al. [226] developed a new brain delivery
carrier, cationic bovine serum albumin (CBSA) conjugated
with PEG-PLA NP (CBSA-NP). CBSA is characterized by
favorable pharmacokinetic properties with a longer serum
half-life and a greater degree of selectivity to brain tissue as
compared to other organs (liver, heart, and lung). Therefore
coupling CBSA, as a brain specific targetor, with PEGylated
NP resulted in an increased NP permeability across the BBB
by absorptive mediated transcytosis, without opening the
endothelial tight junction.
Significant brain uptake of a novel NP formulation,
comprising biocompatible materials such as emulsifying
wax and Brij ® 72 as NP matrix materials, and Brij® 78 and
Tween® 80 as surfactants, respectively, was demonstrated by
using the in-situ rat brain perfusion technique [227, 228].
NP containing Tween® 80 resulted in a statistically
significant increase of brain transport over NP containing
Brij ® 78, in general agreement with Kreuter’s findings
concerning the role of Tween ® 80 in promoting NP brain
uptake. In order to obtain a more specific targeting, the
authors have incorporated thiamine as a surface ligand on the
emulsifying wax and Brij® 78 NP. A comparison of the NP
brain uptake demonstrated that the thiamine-coated NP
associated with the BBB thiamine transporter had an
increased Kin (uptake transfer constant) between 45 and 120
s, indicating that the addition of a thiamine ligand to the NP
is a new, viable approach for NP mediated brain drug
delivery.
On the basis that some naturally occurring peptides can
effectively cross the BBB due to receptor-mediated transport
(transcytosis), Costantino et al. [229] developed an elegant
system, using modified poly(d,l-lactide-co-glycolide)
(PLGA) copolymers obtained by conjugating PLGA and five
short synthetic peptides, which bear some resemblance to the
synthetic opioid peptide MMP-2200 (H2N–L-Tyr-D-Thr-
Gly-L-Phe-L-Leu-L-Ser–O–β-D-lactose–CONH2). The Tyr
present in this peptide was substituted with Phe in order to
avoid a potential opioid effect and the permeability was
supposed to be enhanced by the presence of glycosidic
moieties (glucose, lactose, etc.). The choice of the PLGA as
a copolymer to be modified was due to its biodegradability,
Ricci et al.
the avoidance of inflammatory or immune reaction
induction, FDA status, and its easy covalent linking to
peptides. Fluorescent and confocal microscopy studies
showed that these peptide-derivatized biodegradable NP were
able to cross the BBB, although more studies are needed to
quantify their presence in the CNS and clarify their BBB
crossing mechanisms.
4.4. Solid Lipid Nanoparticles, and Lipid-Drug
Conjugate Nanoparticles
Biodegradable polymers have been widely used for NP
preparation. Although they can meet special technological
and pharmaceutical requirements, thanks to the large
possibilities of structure assemblage, some safety regulatory
problems can derive from the presence of residues of the
organic solvent used, as well as potential polymer
cytotoxicity. Moreover, a scale-up of the NP production
processes can be difficult and therefore, so far, these carrier
systems have not been considered by the pharmaceutical
companies. For all these reasons, increasing attention has
been focused since the beginning of the 1990s on alternative
NP made from solid lipids, the so-called SLN [230]. The
great interest for these carriers resides in the fact that they
combine all the advantages of the traditional colloidal
systems (e.g., physical stability, protection of incorporated
labile drugs from degradation, modified release, excellent
tolerability) while at the same time, avoiding some of their
major disadvantages [231, 232]. Moreover, the lipids used
are well tolerated by the body (e.g., glycerides composed of
fatty acids which are present in the emulsions for parenteral
nutrition) and large scale production can be achieved in a
cost-effective and relatively simple way. Since SLN could be
used for all parenteral applications just as well as polymeric
NP, they have been investigated as drug carriers for brain
delivery. The first promising results were obtained by Yang
et al. [233] with the lipophilic antitumor drug,
camptothecin. Similarly, a significant amount of
doxorubicin in rat brain was reported by Fundarò et al. [234]
after i.v. administration in rats of doxorubicin loaded non-
stealth and stealth SLN. The highest doxorubicin
concentration was however obtained with stealth SLN. Wang
et al. [235] have found enhanced targeting, and subsequent
increased brain uptake of the synthesized more lipophilic
pro-drug 3’,5’-dioctanoyl-5-fluoro-2’-deoxyuridine
(DOFUdR) when incorporated into SLN (DOFUdR-SLN).
This behavior was ascribed to concomitant effects: i) the
modified SLN surface with Pluronic F-68 decreased the
adsorption of plasma opsonins and prolonged the SLN
retention time in plasma; ii) the increased retention of
DOFUdR-SLN in the brain blood capillaries, combined to
the adsorption to the capillary walls, created a higher
concentration gradient and enhanced the transport across the
endothelial cell layer; iii) the possible DOFUdR-SLN
endocytosis by the endothelial cells, followed by the release
of the drug within these cells, delivered the active to the
brain. Successful targeting to the brain was shown by using
Tween 80 stabilized SLN [236], which was well in
agreement with previous studies conducted by Kreuter et al.
[212, 213] on polymeric NP. Due to the SLN low
hydrophilic drug loading capacity, only highly potent
peptides and proteins can be delivered using this approach.
Delivering Drugs to the CNS
In light of this consideration, a new strategy consisting in
drug transformation into water-insoluble lipid-drug
conjugates (LDC) was developed. LDC NP were formed by
high pressure homogenization [237]. LDC-derivatives coated
with Tween® 80 are able to cross the BBB and release the
drug via matrix degradation. LDC NP were used with
diminazene, an active drug against trypanosomiasis, and
were likely to deliver the drug to the brain. ApoE is a key
factor in passing through the BBB, nevertheless Apo A-I
and A-IV were also adsorbed on LDC to prevent their uptake
in the liver prior to their arrival on the BBB ApoE receptors
[238].
4.5. Miscellanea
Other approaches have been developed to overcome BBB
low permeability by means of nanoparticulate drug carrier
strategy. Vinogradov et al. [239] have developed the so-
called nanogels, made from a network of cross-linked ionic
polyethylenimine (PEI) and non-ionic PEG chains (PEG-cl-
PEI). When a drug is associated to the nanogel by
electrostatic interactions, the PEI chains tend to collapse
resulting in the decreased volume and size of the particles.
Due to the steric stabilization of PEG chains a stable
dispersion with a mean particle size of 80 nm can be
obtained. To achieve active targeting, specific ligands can be
covalently attached to free amino groups of PEI fragments,
or to PEI partially modified fragments having biotin
moieties, allowing the attachment of ligand through the
classical biotin-avidin coupling chemistry. Nanogels have
been evaluated as potential carriers for oligonucleotide (ON)
delivery to the brain [240] by using bovine brain
microvessel endothelial cells (BBMEC) as monolayers
mimicking the BBB. In vitro studies have shown an
increased transport of ON across the cell monolayers as a
result of their incorporation into nanogels. Increased ON
transport was observed when the nanogel carriers were
modified with insulin or transferrin ligands [241].
Permeability assays with mannitol indicated that the
increased transport of ON-nanogels did not result from
paracellular diffusion due to a disruption of the BBMEC
monolayers. In vivo experiments in mice showed no adverse
toxic effects of ON-nanogels after intravenous injection,
while increased brain and decreased liver/spleen
accumulations were observed suggesting that this system is
good for the ON delivery to the brain.
An emerging and very interesting strategy for enhancing
drug delivery to the CNS is the use of self-assembling
amphiphilic block copolymers. Individual copolymer
strands, unimers, tend to organize at the critical micellar
concentration (CMC), in micelles which have been proved to
be very promising carriers especially for poorly soluble
drugs [242]. Kabanov et al. [243] had shown, at the
beginning of the 1990s, that Pluronic P85 micelles
(containing two exterior hydrophilic POE blocks and an
interior POP block), conjugated with antibodies, provided
an effective transport of the solubilized neuroleptic agent
haloperidol across the BBB, responsible for its strong
efficacy improvement. However, more recent studies [244]
have demonstrated that the remarkable enhancement of drug
cell penetration in BBMEC monolayers can be ascribed
mainly to Pluronic unimers through the inhibition of the P-
Current Medicinal Chemistry, 2006 Vol. 13, No. 15 1771
gp mediated drug efflux system. Witt et al. [245] have
obtained similar results by investigating the analgesic effect
of enkephalin and biphalin when co-administered with
Pluronic P85 above (1.0%) and below (0.01%) the CMC. In
fact, they have shown an increased analgesic effect of these
drugs in the presence of a Pluronic P85 concentration of
0.01%, whereas a lower analgesia was observed at a higher
Pluronic P85 concentration (0.1%) because of the micellar
trapping which reduced the free drug amount available for
transcellular flux. Although colloidal carriers are ideal for
brain drug targeting for the reasons described above, the
major persisting problem is the limited drug which is due to
the reduced carrier size and to the physico-chemical
properties of the raw materials used for their preparation.
Therefore, considering these drawbacks, a new approach for a
specific targeting of poorly soluble molecules, is to use the
drug itself to form suitable drug NP or nanocrystals
(DissoCubes®, Nanopure®) [246]. Similarly to other
nanoparticulate systems, parenteral administration of
nanocrystals requires a proper modification of their surface
either to avoid the RES or to selectively target the drug to
the brain. In this regard, the type of surfactant was shown to
influence the passage of atovaquone through the BBB for
toxoplasma gondii infections treatment [247]. Again,
Tween® 80 was used to modify the nanocrystal surface
leading to in vivo preferential adsorption of ApoE to achieve
brain drug therapeutic concentration levels.
CONCLUDING REMARKS
Over the last decade, medicinal chemistry and
pharmaceutical technology have been following divergent
routes to try to solve the crucial issues related to the
development of effective CNS targeting strategies.
Historically, medicinal chemistry competences have focused
on the selection of more potent and selective drug
candidates, whereas pharmaceutical technology has mainly
been dedicated to solving ADME issues [51]. In the 1990s,
mostly due to economic reasons, companies were forced to
start investigating critical pharmacokinetic characteristics at
the early stages of the drug discovery process [51]. At this
point, the development of new in vitro and in silico
prediction models was implemented. This new approach
aided the selection of the best candidates on the basis of in
vitro high throughput screenings. Similarly, CNS drug
discovery programs advanced accordingly. Unfortunately, the
underestimation and lack of knowledge of the mechanisms
governing CNS uptake led to the exclusion of most
potential neurotherapeutics, which did not match with these
poorly established criteria. Moreover, at the present day, in
the industry, valid BBB programs are virtually yet non-
existent. This failure should push researchers toward another
direction, especially since the parallel increased knowledges
in pharmaceutical technology and progress in BBB function
acquaintances have actively contributed to the development
and improvement of new strategies for CNS targeting.
In light of these considerations, the question concerning
whether or not drug delivery to the CNS is a medicinal
chemistry or a pharmaceutical technology issue, intended
principally to solve BBB problems, can be partially
answered in favor of the pharmaceutical technology [2, 3]. In
1772 Current Medicinal Chemistry, 2006, Vol. 13, No. 15 Ricci et al.

fact, considering also the future research trends (genomics, [22] Garberg, P. ATLA-Altern. Lab. Anim., 1998, 26, 821.
[23] Lundquist, S.; Renfte, M. Vasc. Pharmacol. , 2002, 38, 355.
proteomics), the development of therapeutic molecules will [24] Balabanov, R.; Dore-Duffy, P. J. Neurosci. Res., 1998, 53, 637.
shift inevitably to the identification of new potential CNS [25] Kondo, A.; Inoue, T.; Zagara, H.; Tateishi, J.; Fukui, M. Brain
drug candidates, consisting mainly in large molecules, such Res., 1987, 412, 73.
as peptides, proteins and nucleic acids that unfortunately are [26] Lo, E.H., Singhal, A.B.; Torchilin, V.P.; Abbott, N.J. Brain Res.
unable to cross the BBB [3, 6]. Therefore, the pharma- Rev., 2001, 38, 140.
[27] Gaetz, M. Clin. Neurophysiol., 2004, 115, 4.
ceutical technology contribution could ease the frustration [28] Krewson, C.E.; Klarman, M.L.; Saltzman W.M. Brain Res., 1995,
caused by the restricted possibility of using the huge number 680, 196.
of specific powerful drug candidates that are theoretically [29] Menei, P.; Pean, J.M.; Nerrière-Daguin, V.; Jollivet, C.; Brachet,
available. All this, will force medicinal chemistry and P.; Benoit, J. P. Exp. Neurol., 2000, 161, 259.
[30] Anderson, K.D.; Anderson, R.F.; Altar C.A.; DiStefano P.S.;
pharmaceutical technology to work more closely to achieve a Corcoran T.L.; Lindsay, R.M.; Wiegand, S.J. Soc. Neurosci.
common goal: the optimization of CNS disease care. Abstr., 1993, 19, 662.
Furthermore, this new synergy may mark the passage from a [31] Guerin, C.; Olivi, A.; Weingart, J.D.; Lawson, C.H.; Brem, H.
molecular ADME to a supramolecular ADME approach. Invest. New Drug, 2004, 22, 27.
Hence, a common converging path is now being drawn by [32] Wang, P.P.; Frazier, J.; Brem, H. Adv. Drug Deliv. Rev., 2002, 54,
987.
proteomics and gene therapy, which leads to a [33] Yan, Q.; Matheson, C.; Sun, J.; Radeke, M.J.; Feinstein, S.C.;
reconsideration of the basic beliefs and assumptions Miller, J.A. Exp. Neurol., 1994, 127, 23.
regarding both fields. [34] Bar, T. Adv. Anat. Embriol. Cell Biol., 1980, 59, 1.
[35] Illum, L. Eur. J. Pharm. Sci., 2000, 11, 1.
In this context, pharmaceutical technology will actively [36] Graff, C.L.; Pollack, G.M. J. Pharm Sci. , 2005, 94, 1187.
contribute to gene medicine for the successful delivery of the [37] Vyas, T.K.; Babbar, A.K.; Sharma, R.K.; Singh, S.; Misra, A.
drugs to the CNS, not only by circumventing the BBB, but AAPS Pharm. Sci. Tech., 2006, 7 (1) Article 8.
[38] Kao, H.D.; Traboulsi, A.; Itoh, S.; Dittert, L.; Hussain, A. Pharm.
also for the drug targeting to the right site and for the needed Res., 2000, 17, 978.
period of time. In this scenario the borderline between [39] Borlongan, C.V.; Emerich, D.F. Brain Res. Bull., 2003, 60, 297.
medicinal chemistry and pharmaceutical technology [40] Kemper, E.A.; Boogerd, W.; Thuis, I.; Beijnen, J.H.; Tellinger,
competences will probably fade. O.V. Cancer Treat. Rev., 2004, 30, 415.
[41] Grieb, P.; Forster, R.E.; Strome, D.; Goodwin, C.W.; Pape, P.C. J.
Are we going towards a “medicinal technology”? Appl. Physiol., 1985, 58, 1929.
[42] Ramsay, R.E.; Hammond, E.J.; Perchalski, R.J.; Wilder, B.J. Arch.
Neurol., 1979, 36, 535.
REFERENCES [43] Habgood, M.D.; Begley, D.J.; Abbott, N.J. Cell. Mol. Neurobiol.,
2000, 20, 231.
[1] Olesen, J.; Leonardi, M. Eur. J. Neurol., 2003, 10, 471. [44] Levin, V.A.; J. Med. Chem., 1980, 23, 682.
[2] Pardridge, W.M. Drug Discov. Today , 2002, 7, 5. [45] Jezequel, S.G. Prog. Drug Metab., 1997, 13, 132.
[3] Lawrence, R.N. Drug Discov. Today , 2002, 7, 223. [46] Smith, Q.R. Int. Congr. Series, 2005, 1277, 63.
[4] Abbott, N.J. Neurochem. Int., 2004, 45, 545. [47] Greig, N.H.; Genka, S.; Daly, E.M.; Sweeney, D.J.; Rapoport, S.I.
[5] Ghersi-Egea, J.F.; Sugiyama, Y. Adv. Drug Deliver. Rev., 2004, Cancer Chemiother. Pharmacol., 1990, 13, 132.
56, 1693. [48] Greig, N.H.; Daly, E.M.; Sweeney, D.J.; Rapoport, S.I. Cancer
[6] Pardridge, W.M. Brain Drug Targeting: The Future of Brain Drug Chemiother. Pharmacol., 1990, 25, 320.
Development, Cambridge University Press: Cambridge U.K., [49] Young, R.C.; Mitchell, R.C.; Brown, T.H.; Ganellin, C.R.;
2001. Griffiths, C.R.; Jones, M.; Rana, K.K.; Saunders, D. J. Med.
[7] Pardridge, W.M. Introduction to the Blood-Brain Barrier: Chem., 1988, 31, 656.
Methodology, Biology and Pathology, Cambridge University Press: [50] Seelig, A.; Gottschlich, R.; Dervent, R.M. P. Natl. Acad. Sci. USA,
Cambridge U.K. 1998. 1994, 91, 68.
[8] Copin, J.C.; Gasche, Y. Ann. Fr. Anesth., 2003, 22, 202. [51] Clark, D.E. Drug Discov. Today , 2003, 20, 927.
[9] Ballabh, P.; Braun, A.; Nedergaard, M. Neurobiol. Dis., 2004, 16, [52] Abbott, N.J.; Drug Discov. Today , 2004, 1, 407.
1. [53] De Sarro, A.; Cecchetti, V.; Fravolini, A.; Naccari, F.; Tabarrini,
[10] Matter, K.; Balda, M.S. Methods, 2003, 30, 228. O. Antimicrob. Agents Ch., 1999, 43, 1729.
[11] Lapierre, L.A. Adv. Drug Deliver. Rev., 2000, 41, 255. [54] Qutub, A.A.; Hunt, C.A. Brain Res. Rev., 2005, 49, 595.
[12] Kniesel, U.; Wolburg, H. Cell. Mol. Neurobiol., 2000, 20, 57. [55] McEwen, B.S.; Reagan, L.P. Eur. J. Pharmacol. , 2004, 49, 13.
[13] Wolburg, H.; Lippoldt, A. Vasc. Pharmacol. , 2002, 38, 323. [56] Cornford, E.M.; Hyman, S.; Swartz, B.E. J. Cereb. Blood Flow
[14] Gee, J.R.; Keller, J.N. Int. J. Biochem. Cell B., 2005, 37, 1145. Metab., 1994, 14, 106.
[15] Aschner, M. Toxicol. Lett., 1998, 102-103, 283. [57] Bonina, F.P.; Arenare, L.; Palagiano, F.; Saija, A.; Nava, F.;
[16] Tao-Cheng, J.H.; Nagy, Z.; Brightman, M.W. J. Neurosci., 1987, Trombetta, D.; de Caprariis, P. J. Pharm. Sci. , 1999, 88, 561.
7, 3293. [58] Bonina, F.P.; Arenare, L.; Ippolito, R.; Boatto, G.; Battaglia, G.;
[17] Isobe, I.; Watanabe, T.; Yotsuyanagi, T.; Hazemoto, N.; Bruno, V.; de Caprariis, P. Int. J. Pharm., 2000, 202, 79.
Yamagata, K.; Ueki, T.; Nakanishi, K.; Asai, K.; Kato, T. [59] Stone, T.W. Prog. Neurobiol., 2001, 64, 185.
Neurochem. Int., 1996, 28, 523. [60] Stone, T.W. Trends Pharmacol. Sci., 2000, 21, 149.
[18] Panzer, R.C.; Raff, M.C. Nature, 1987, 325, 253. [61] Tamai, I.; Tsuji, A. J. Pharm. Sci. , 2000, 89, 1371.
[19] Neuhaus, J.; Risau, W.; Wolburg, H. Ann. N.Y. Acad. Sci., 1991, [62] Kanai, Y.; Segawa, H.; Miyamoto, K.; Uchino, H.; Takeda, E.;
633, 578. Endou, H. J. Biol. Chem., 1998, 273, 23629.
[20] Rubin, L.L.; Hall, D.E.; Porter, S.; Barbu, K.; Cannon, C.; Horner, [63] Uchino, H.; Kanai, Y.; Kim, D.K.; Wempe, M.F.; Chairoungdua,
H.C.; Janatpour, M.; Liaw, C.W.; Manning, K.; Morales, J.; A.; Morimoto, E.; Anders, M.W.; Endou, H. Mol. Pharmacol.,
Tanner, L.I.; Tomaselli, K.J.; Bard, F. J. Cell Biol., 1991, 115, 2002, 61, 729.
1725. [64] Walzer, J.; Nicholls, D.; Irwin, W.J.; Freeman, S. Int. J. Pharm.,
[21] Polli, J.W.; Humphrey, J.E.; Wring, S.A.; Burnette, T.C.; Read, 1994, 104, 157.
K.C.; Hersey, A.; Butina, D.; Bertolotti, L.; Pugnaghi, P.; Serabjit- [65] Taylor, C.P.; Gee, N.S.; Su, T.; Kocsis, J.D.; Welty, D.F.; Brown,
Singh, C.J. In Progress in the Reduction, Refinement and J.P.; Dooley, D.J.; Boden, P.; Singh, L. Epilepsy Res., 1998, 29,
Replacement of Animal Experimentation; Balls M, van Zeller A- 233.
M, and Halder M, Eds.; Elsevier Science: New York, 2000; pp. [66] Rice, M.E. Trends Neurosci., 2000, 23, 209.
271–289.
Delivering Drugs to the CNS Current Medicinal Chemistry, 2006 Vol. 13, No. 15 1773

[67] Manfredini, S.; Vertuani, S.; Pavan, B.; Vitali, F.; Scaglianti, M.; [110] Coloma, M.J.; Lee, H.J.; Kurihara, A.; Landaw, E.M.; Boado, R.J.;
Bortolotti, F.; Biondi, C.; Scatturin, A.; Prasad, P.; Dalpiaz, A. Morrison, S.L.; Pardridge, W.M. Pharm. Res., 2000, 17, 2000.
Bioorgan. Med. Chem. , 2004, 12, 5453. [111] Bodor, N.; Prokai, L.; Wu, W.M.; Farag, H.; Jonalagadda, S.;
[68] Dalpiaz, A.; Pavan, B.; Vertuani, S.; Vitali, F.; Scaglianti, M.; Kawamura, M.; Simpkins, J. Science, 1992, 257, 1698.
Bortolotti, F.; Biondi, C.; Scatturin, A.; Manganelli, S.; Ferraro, L.; [112] Prokai, L.; Ouyang, X.; Wu, W.M.; Bodor, N. J. Am. Chem. Soc.,
Marzola, G.; Prasad, P.; Manfredini, S. Eur. J. Pharm. Sci., 2005, 1994, 116, 2643.
24, 259. [113] Prokai, L.; Ouyang, X.; ProkaiTatrai, K.; Simpkins, J.W.; Bodor,
[69] Takanaga, H.; Tamai, I.; Inaba, S.; Sai, Y.; Higashida, H.; N. Eur. J. Med. Chem., 1998, 33, 879.
Yamamoto, H.; Tsuji, A. Biochem. Biophys. Res. Commun., 1995, [114] Prokai, L.; ProkaiTatrai, K.; Ouyang, X.; Kim, H.S.; Wu,. W.M.;
217, 370. Zharikova, A.; Bodor, N. J. Med. Chem., 1999, 42, 4563.
[70] Mackey, J.R.; Mani, R.S.; Selner, M.; Mowles, D.; Young, J.D.; [115] Yoon, S-H.; Wu J.; Wu W.M.; Prokai L.; Bodor N. Bioorg. Med.
Belt, J.A.; Crawford, C.R.; Cass, C.E. Cancer Res. , 1998, 58, 4349. Chem., 2000, 8,1059.
[71] Tsuji, A.; Tamai, I. Adv. Drug Deliv. Rev., 1997, 25, 287. [116] Chen, P.; Bodor, N.; Wu, W.M.; Prokai, L. J. Med. Chem., 1998,
[72] Schinkel, A.H. Adv. Drug Deliv. Rev., 1999, 36, 179. 41, 3773.
[73] Stouch, T.R.; Gudmundsson, O. Adv. Drug Deliv. Rev., 2002, 54, [117] Ganguli, M.; Burmeister, L.A.; Seaberg, E.C.; Belle, S.; DeKosky,
315. S.T. Biol. Psychiat, 1996, 40, 714.
[74] Kusuhara, H.; Sugiyama Y. Drug Discov. Today , 2001, 6, 150. [118] Prokai, L.; Zharikova, A.D. Brain Res., 2002, 952, 268.
[75] Kusuhara, H.; Sugiyama, Y. Drug Discov. Today , 2001, 6, 206. [119] Anholt, R.R.H.; De Souza, E.B.; Oster-Granite, M.L.; Snyder, S.H.
[76] Begley, D.J.; Bradbury, M.W.; Kreuter, J. The Blood-Brain J. Pharmacol. Exp. Ther., 1985, 233, 517.
Barrier and Drug Delivery to the CNS , Marcel Dekker, Inc., New [120] Anholt, R.R.H.; Pedersen, P.L.; De Souza, E.B.; Snyder, S.H. J.
York, 2000. Biol. Chem., 1986, 261, 576.
[77] Sarkadi, B.; Müller, M. Semin. Cancer Biol., 1997, 8, 171. [121] Trapani, G.; La quintana, V.; Latrofa, A.; Ma, J.; Reed, K.; Serra,
[78] Mahar Doan, K.M.; Humphreys, J.E.; Webster, L.O.; Wring, S.A.; M.; Bigio, G.; Liso, G.; Gallo, J.M. Bioconjug. Chem., 2003, 14,
Shampine, L.J.; Serabjit-Singh, C.J.; Adkison, K.K.; Pollithe, J.W. 830.
J. Pharmacol. Exp. Ther., 2002, 303, 1029. [122] Song, B.W.; Vinters, H.V.; Wu, D.; Pardridge, W.M. J.
[79] Polli, J.W.; Wring, S.A.; Humphreys, J.E.; Huang, L.; Morgan, Pharmacol. Exp. Ther. , 2002, 301, 605.
J.B.; Webster, L.O.; Serabjit-Singh, C.J. J. Pharmacol. Exp. Ther., [123] Fleming, A.B.; Haverstick, K.; Saltzman, W.M. Bioconjug. Chem.,
2001, 299, 620. 2004, 15, 1364.
[80] Pardridge, W.M.; Eisenberg, J.;Yang, J. J. Neurochem., 1985, 44, [124] Visser, C.C.; Voorwinden, L.H.; Crommelin, D.J.A.; Danhof, M.;
1771. de Boer, A.G. Pharm. Res., 2004, 21, 756.
[81] Duffy, K.R.; Pardridge, W.M. Brain Res., 1987, 420, 32. [125] Mueller, A.M.; Pedre´, X.; Kleiter, I.; Hornberg, M.;
[82] Pardridge, W.M.; Eisenberg, J.; Yang, J. Metabolism, 1987, 36, Steinbrecher, A.; Giegerich, G. J. Neuroimmunol., 2005, 159, 55.
892. [126] Mohler, H.; Rudolph, U. Drug Discov. Today: Therapeutic
[83] Fishman, J.B.; Rubin, J.B.; Handrahan, J.V.; Connor, J.R.; Fine, Strategies, 2004, 1, 117.
R.E. J. Neurosci. Res., 1987, 18, 299. [127] Resnick, L.; Fennell, M. Drug Discov. Today , 2004, 9, 932.
[84] Reinhardt, R.R.; Bondy, C.A. Endocrinology, 1994, 135, 1753. [128] Chung, N.S.; Wasan, K.M. Adv. Drug Deliv. Rev., 2004, 56, 1315.
[85] Golden, P.L.; Maccagnan, T.J.; Pardridge, W.M. J. Clin. Invest., [129] Zamecnik, P.; Stephenson, M. Proc. Natl. Acad. Sci. USA, 1978,
1997, 99, 14. 75, 280.
[86] Anderson, B.D. Adv. Drug Deliv. Rev., 1996, 19, 171. [130] Van Oekelen, D.; Luyten, W.H.M.L.; Leysen, J.E. Brain Res.
[87] Abbott, J.N.; Romero, I.A. Molec. Med. Today, 1996, 2, 106. Rev., 2003, 42, 123.
[88] Hoste, K.; De Winne, K.; Schacht, E. Int. J. Pharm., 2004, 277, [131] Da Ros, T.; Spalluto, G.; Prato, M.; Saison-Behemoaras, T.;
119. Boutorine, A.; Cacciari, B. Curr. Med. Chem., 2005, 12, 71.
[89] Carelli, V.; Liberatore, F.; Scipione, L.; Impicciatore, M.; [132] Lzsch, B.T.; Wood M. J. Neurochem., 2004, 88, 257.
Barocelli, E.; Cardellini, M.; Giorgioni, G. J. Control. Rel., 1996, [133] Lamensdorf, I. PCT Int. Appl., 2005, 46.
42, 209. [134] Reineke, T.M. PCT Int. Appl., 2005, 147.
[90] Ishikura, T.; Senou, T.; Ishikara, H.; Kato, T.; Ito, T. Int. J. [135] During, M.J.; Vezzani, A., PCT Int. Appl., 2005, 78.
Pharm., 1996, 116, 51. [136] Francis, J.W.; Brown, R.H. Jr.; Fishman, P.S. PCT Int. Appl., 2004,
[91] Jatzkewitz, H. Z. Naturforsch., 1955, 10b, 27. 46.
[92] Panarin, E.F.; Ushakov, S.N. Khim. Pharm. Zhur., 1968, 2, 28. [137] Lamensdorf, I.; Katzhendler, J. PCT Int. Appl., 2005, 35.
[93] Maeda, M.; Izuno, Y.; Kawasaki, K.; Kaneda, Y.; Mu, Y.; [138] Hagstrom, J.E.; Wolff, J.A.; Monahan, S.D.; Rozema, D.B.;
Tsutsumi, Y.; Nakagawa, S.; Mayumi, T. Chem. Pharm. Bull., Budker, V.G.; Slattum, P.M.; Lewis, D.L. U.S. Pat. Appl. Publ.,
1997, 45, 1788. 2004, 35.
[94] Seymour, L.W.; Ulbrich, K.; Strohalm, J.; Duncan, R. Biochem. [139] Lamensdorf, I.; Katzhendler, J. PCT Int. Appl., 2005, 30.
Pharmacol., 1990, 39, 1125. [140] Feld, Y.; Gepstein, L.; Marom, S. U.S. Pat. Appl. Publ., 2005, 57.
[95] Borlongan, C.V.; Emerich, D.F. Brain Res. Bull., 2003, 60, 297. [141] Zankel, T.; Starr, C.M.; Gabathuler, R. PCT Int. Appl., 2005, 192.
[96] Bodor, N.; Buchwald, P. Adv. Drug Deliv. Rev., 1999, 36, 229. [142] Byrnes, A.; Rusby, J.; Wood, M.; Charlton, H. Neuroscience,
[97] Bodor, N.; Brewster, M.E. Pharmacol. Ther., 1982, 19, 337. 1995, 66, 1015.
[98] Bodor, N.; Brewster, M.E. In Targeted Drug Delivery; Juliano, [143] Braasch, D.; Jensen, S.Y.L.; Liu, Y.; Kaur, K.; Arar, K.; White,
R.L. Ed.; Springer Eds., Berlin, 1991; pp. 231–284. M.A.; Corey D.R. Biochemistry, 2003, 42, 7964.
[99] Bodor, N.; Buchwald, P. Drug Discov. Today , 2002, 14, 766. [144] Sun, L.Q.; Cairns, M.J.; Saravolac, E.G.; Baker, A.; Gerlach,
[100] Somogyi, G.; Nishitani, S.; Nomi, D.; Buchwald, P.; Prokai, L.; W.L. Pharmacol. Rev., 2000, 52, 325.
Bodor, N. Int. J. Pharm., 1998, 166, 15. [145] Vester, B.; Lundberg, L.B.; Sorensen, M.D.; Babu, B.R.;
[101] Somogyi, G.; Buchwald, P.; Nomi, D.; Prokai, L.; Bodor, N. Int. J. Douthwaite, S.; Wengel, J. J. Am. Chem. Soc., 2002, 124, 13682.
Pharm., 1998, 166, 27. [146] Parry, T.J.; Cushman, C.; Gallegos, A.M.; Agrawal, A.B.;
[102] Borlongan, C.V.; Emerich, D.F. Brain Res. Bull., 2003, 60, 297. Richardson, M.; Andrews, L.E.; Maloney, L.; Mokler, V.R.;
[103] Straub, J.A.; Akiyama, A.; Parmar, P. Pharm. Res., 1994, 11, Wincott, F.E.; Pavco, P.A. Nucleic Acids Res., 1999, 27, 2569.
1673. [147] LaVail, M.M.; Yasumura, D.; Matthes, M.T.; Drenser, K.A.;
[104] Kemper, E.M.; Verheij, M.; Boogerd, W.; Beijnen, J.H.; van Flannery, J.G.; Lewin, A.S.; Hauswirth, W.W. P. Natl. Acad. Sci.
Tellingen, O. Eur. J. Cancer , 2004, 40, 1269. USA, 2000, 97, 11488.
[105] Bodor, N.; Buchwald, P. Chem. Br., 1998, 34, 36. [148] Xia, H.; Mao, Q.; Paulson, H.L.; Davidson, B.L. Nat. Biotechnol.,
[106] Wu, D.; Yang, J.; Pardridge, W.M. J. Clin. Invest., 1997, 100, 2002, 20, 1006.
1804. [149] Rubinson, D.A.; Dillon, C.P.; Kwiatkowski, A.V.; Sievers, C.;
[107] Kurihara, A.; Deguchi, Y.; Pardridge, W.M. Bioconjugate Chem., Yang, L.; Kopinja, J.; Rooney, D.L.; Ihrig, M.M.; McManus, M.T.;
1999, 10, 502. Gertler, F.B.; Scott, M.L.; Van Parijs, L. Nat. Genet., 2003, 34,
[108] Wu, D.; Pardridge, W.M. J. Pharmacol. Exp. Ther., 1996, 279, 77. 231.
[109] Wu, D.; Pardridge, W.M. P. Natl. Acad. Sci. USA, 1999, 96, 254. [150] Jin, J.Y.; Moon, C.I.; Kang, W.S.; Jeong; D.C. J. Korean Med.
Sci., 2003, 18, 108.
1774 Current Medicinal Chemistry, 2006, Vol. 13, No. 15 Ricci et al.

[151] Alyautdin, R.; Gothier, D.; Petrov, V.; Kharkevich, D.; Kreuter, J. [188] Kang, Y.S.; Bickel, U.; Pardridge, W.M. Drug Metab. Dispos.,
Eur. J. Pharm. Biopharm., 1995, 41, 44. 1994, 22, 99.
[152] Kreuter, J.; Alyautdin, R.N.; Kharkevich, D.A.; Ivanov, A.A. [189] Pardridge, W.M.; Boado, R.J.; Kang, Y.S. P. Natl. Acad. Sci. USA,
Brain Res., 1995, 674, 171. 1995, 92, 5592.
[153] Schroder, V.; Sabel, B.A. Brain Res., 1996, 710, 121. [190] Shi, N.; Boado, R.J.; Pardridge, W.M. Pharm. Res., 2000, 118,
[154] Gulyaev, A.E.; Gelperina, S.E.; Skidan, I.N.; Antropov, A.S.; 1095.
Kivman, G.Y.; Kreuter, J. Pharm. Res., 1999, 16, 1564. [191] Zhu, C.; Zhang, Y.; Zhang, Y.F.; Yi Li, J.; Boado, R.J.; Pardridge,
[155] Cheng, Y.S.; Fang, L.L.; Ying, C.; Bi, Z.J.; Wen, L.B.; Zheng, Y.C. W.M. J. Gene Med., 2004, 6, 906.
J. Control. Release, 1999, 59, 299. [192] Schlachetzki, F.; Zhang, Y.F.; Boado, R.J.; Pardridge, W.M.
[156] Moghimi, S.M.; Hunter, A.C.; Murray, J.C. Pharmcol. Rev., 2001, Neurology, 2004, 62, 1275.
53, 283. [193] Boado, R.J. NeuroRx, 2005, 2, 139.
[157] Ogawara, K.; Furumoto, K.; Takakura, Y.; Hashida, M.; Higaki, [194] Mamot, C.; Drummond, D.C.; Greiser, U.; Hong, K.; Kirpotin,
K.; Kimura T. J. Control. Release, 2001, 77, 191. D.B.; Marks, J.D.; Park, J.W. Cancer Res. , 2003, 63, 3154.
[158] Furumoto, K.; Nagayama, S.; Ogawara, K.; Takakura, Y.; [195] Pardridge, W.M. Nat. Rev. Drug. Discov., 2002, 1, 131.
Hashida, M.; Higaki, K.; Kimura, T. J. Control. Release, 2004, 97, [196] Zhang, Y.F.; Zhu, C.; Pardridge, W.M. Mol. Ther., 2002, 6, 67.
133. [197] Zhang, Y.F.; Boado, R.J.; Pardridge, W.M. Pharm. Res., 2003, 20,
[159] Peracchia, M.T.; Vauthier, C.; Desmaele, D.; Gulik, A.; Dedieu, 1779.
J.C.; Demoy, M.; d’Angelo, J.; Couvreur, P. Pharm. Res., 1998, [198] da Cruz, M.T.; Simoes, S.; de Lima, M.C. Exp. Neurol., 2004, 187,
15, 550. 65.
[160] Peracchia, M.T.; Fattal, E.; Desmaele, D.; Besnard, M.; Noel, J.P.; [199] Pardridge, W.M. Neuron, 2002, 36, 555.
Gomis, J.M.; Appel, M.; d’Angelo, J.; Couvreur, P. J. Control. [200] Garcia-garcia, E.; Andrieux, K.; Gil, S.; Couvreur, P. Int. J.
Release, 1999, 60, 121. Pharm., 2005, 298, 274.
[161] Moghimi, S.M.; Patel, H.M. Biochim. Biophys. Acta, 1992, 1135, [201] Yoshida, J.; Mizuno, M. J. Neuro-Oncol., 2003, 65, 261.
269. [202] Yoshida, J.; Mizuno, M.; Fujii, M.; Kajita, Y.; Nakahara, N.;
[162] Huwyler, J.; Wu, D.; Pardridge, W.M. P. Natl. Acad. Sci. USA, Hatano, M.; Saito, R.; Nobayashi, M.; Wakabayashi, T. Hum.
1996, 93, 14164. Gene Ther., 2004, 15, 77.
[163] Frank, M.M. J. Lab. Clin. Med., 1993, 122, 487. [203] Mizuno, M.; Ryuke, Y.; Yoshida, J. Cancer Gene Ther., 2002, 9,
[164] Gabizon, A.; Shmeeda, H.; Barenholz, Y. Clin. Pharmacokinet., 825.
2003, 42, 419. [204] Voges, J.; Weber, F.; Reszka, R.; Sturm, V.; Jacobs, A.; Heiss,
[165] Uster, P.S.; Allen, T.M.; Daniel, B.E.; Mendez, C.J.; Newman, W.D.; Wiestler, O.; Kapp, J.F. Hum. Gene Ther., 2002, 13, 675.
M.S.; Zhu, G.Z. FEBS Lett., 1996, 386, 243. [205] Ishida, T.; Ichikawa, T.; Ichihara, M.; Sadzuka, Y.; Kiwada, H. J.
[166] Papahadjopoulos, D.;. Allen, T.M.; Gabizon, A.; Mayhew, M E.; Control. Release, 2004, 95, 403.
Matthay, K.; Huang, S.K.; Lee, K.; Woodle, M.C.; Lasic, D.D.; [206] Ishida, T.; Harada, M.; Wang, X.-Y.; Ichihara, M.; Irimura, K.;
Redemann, C.; Martin, F.J. P. Natl. Acad. Sci. USA, 1991, 88, Kiwada, H. J. Control. Release, 2005, 105, 305.
11460. [207] Kreuter, J. In Colloidal Drug Delivery Systems; J. Kreuter, Ed.,
[167] Ishida, T.; Harashima, H.; Kiwada, H. Biosci. Rep., 2002, 22, 197. Marcel Dekker: New York, 1994; Vol. 66, pp. 219–342.
[168] Ceh, B.; Winterhalter, M.; Frederik, P.M.; Vallner, J.J.; Lasic, [208] Bodmeier, R.; Maincent, P. In Pharmaceutical dosage forms:
D.D. Adv. Drug Deliv. Rev., 1997, 24, 165. disperse systems H.A. Lieberman, M.M. Rieger and G.S. Banker
[169] Allen, T.M. Trends Pharmacol. Sci., 1994, 15, 215. Eds.; Marcel Dekker: New York, 1998, Vol. 3, pp. 87-127.
[170] Hau, P.; Fabel, K.; Baumgart, U.; Rummele, P.; Grauer, O.; Bock, [209] Couvreur, P.; Dubernet, C.; Puisieux, F. Eur. J. Pharm. Biopharm.
A.; Dietmaier, C.; Dietmaier, W.; Dietrich, J.; Dudel, C.; Hubner, 1995, 41, 2.
F.; Jauch, T.; Drechsel, E.; Kleiter, I.; Wismeth, C.; Zellner, A.; [210] Grislain, L.; Couvreur, P.; Lenaerts, V.; Roland, M.; Deprez-
Brawanski, A.; Steinbrecher, A.; Marienhagen, J.; Bogdahn, U. Decampeneere, D.; Speiser, P. Int. J. Pharm., 1983, 15, 335.
Cancer, 2004, 100, 1199. [211] Kreuter, J.; Petrov, V.E.; Kharkevich, D.A.; Alyautdin, R.N. J.
[171] Sharpe, M.; Easthope, S.E.; Keating, G.M.; Lamb, H.M. Drugs, Control. Release, 1997, 49, 81.
2002, 62, 2089. [212] Kreuter, J. Adv. Drug Deliv. Rev., 2001, 47, 65.
[172] Koukourakis, M.I.; Koukouraki, S.; Giatromanolaki, A.; [213] Kreuter, J. Pharm. Acta Helv., 1983, 58, 242.
Kakolyris, S.; Georgoulias, V.; Velidaki, A.; Archimandritis, S.; [214] Schroeder, U.; Schroeder, H.; Sabel, B.A. Life Sci., 2000, 66, 495.
Karkavitsas, N.N. Acta Oncol., 2000, 39, 207. [215] Alyautdin, R.N.; Petrov, V.E.; Langer, K.; Berthold, A.;
[173] Koukourakis, M.I.; Koukouraki, S.; Fezoulidis, I.; Kelekis, N.; Kharkevich, D.A.; Kreuter, J. Pharm. Res., 1997, 14, 325.
Kyrias, G.; Archimandritis, S.; Karkavitsas, N. Brit. J. Cancer, [216] Alyautdin, R.N.; Tezikov, E.B.; Ramge, P.; Kharkevich, D.A.;
2000, 83, 1281. Begley, D.J.; Kreuter, J. J. Microencapsul., 1998, 15, 67.
[174] Schmidt, J.; Metselaar, J.M.; Wauben, M.H.; Toyka, K.V.; Storm, [217] Friese, A.; Seiller, E.; Quack, G.; Lorenz, B.; Kreuter, J. Eur. J.
G.; Gold, R. Brain, 2003, 126, 1895. Pharm. Biopharm., 2000, 49, 103.
[175] Zhang, Y.; Zhang, Y.-F.; Bryant, J.; Charles, A.; Boado, R.J.; [218] Gulyaev, A.E.; Gelperina, S.E.; Skida, A.S.; Antropov, G.Y.;
Pardridge, W.M. Clin. Cancer Res., 2004, 10, 3667. Kreuter, J. Pharm. Res., 1999, 16, 1564.
[176] Pardridge, W.M. Adv. Drug Deliv. Rev., 1995, 15, 109. [219] Steiniger, S.C.J.; Kreuter, J.; Khalansky, A.S.; Skidan, I.N.;
[177] Pardridge, W.M., Drug Deliv., 1993, 1, 83. Bobruskin, A.I.; Smirnova, Z.S.; Severin, S.E.; Uhl, R.; Kock, M.;
[178] Wu, D.; Yang, J.; Pardridge, W.M. J. Clin. Invest., 1997, 100, 180. Geiger, K.D.; Gelperina. S.E. Int. J. Cancer, 2004, 109, 759.
[179] Schnyder, A.; Huwyler, J. NeuroRx, 2005, 2, 99. [220] Kreuter, J. Int. Congress Series, 2005, 1277, 85.
[180] Kaasgaard, T.; Mouritsen, O.G.; Jorgensen. K. Int. J. Pharm., [221] Olivier, J.C.; Fenart, L.; Chauvet, R.; Pariat, C.; Cecchelli, R.;
2001, 214, 63. Couet, W. Pharm. Res., 1999, 16, 1836.
[181] Mori, A.; Klibanov, A.L.; Torchilin, V.P.; Huang, L. FEBS Lett. , [222] Kreuter, J.; Ramge, P.; Petrov, V.; Hamm, S.; Gelperina, S.E.;
1991, 284, 263. Engelhardt, B.; Alyautdin, R.; von Briesen, H.; Begley, D.J.
[182] Maruyama, K.; Takizawa, T.; Yuda, T.; Kennel, S.J.; Huang, Pharm. Res., 2003, 20, 409.
L.M. Iwatsuru, M. Biochim. Biophys. Acta , 1995, 1234, 74. [223] Kreuter, J.; Shamenkov, D.; Petrov, V.; Ramge, P.; Cychutek, K.;
[183] Allen, T.M.; Brandeis, E.; Hansen, C.B.; Kao, G.Y.; Zalipsky, S. Koch-Brandt, C.; Alyautdin, R. J. Drug Target, 2002, 10, 317.
Biochim. Biophys. Acta , 1995, 1237, 99. [224] Olivier, J.C.; Huerta, R.; Lee, H.J.; Calon, F.; Pardridge, W.M.
[184] Bendas, G.; Krause, A.; Bakowsky, U.; Vogel, J.; Rothe, U. Int. J. Pharm. Res., 2002, 19, 117.
Pharm., 1999, 181, 79. [225] Aktas, Y.; Yemisci, M.; Andrieux, K.; Gursoy, R. N.; Alonso,
[185] Shahinian, S.; Silvius, J.R. Biochim. Biophys. Acta, 1995, 1239, M.J.; Fernandez-Megia, E.; Novoa-Carballal, R.; Quiñoa´, E.;
157. Riguera, R.; Sargon, M.F.; Celik, H.H.; Demir, A.S.; Hıncal, A.A.;
[186] Hansen, C.B.; Kao, G.Y.; Moase, E.H.; Zalipsky, S.; Allen, T.M. Dalkara, T.; Capan, Y.; Couvreur, P. Bioconjug. Chem., 2005, 16,
Biochim. Biophys. Acta , 1995, 1239, 133. 1503.
[187] Jefferies, W.A.; Brandon, M.R.; Williams, A.F.; Hunt, S.V. [226] Lu, W.; Tan, Y.-Z.; Hu, K.-L.; Jiang, X.-G. Int. J. Pharm., 2005,
Immunology, 1985, 54, 333. 295, 247.
Delivering Drugs to the CNS Current Medicinal Chemistry, 2006 Vol. 13, No. 15 1775

[227] Lockman, P.R.; Oyewumi, M.O.; Koziara, J.M.; Roder, K.E.; [239] Vinogradov, S.V.; Bronich, T.K.; Kabanov, A.V. Adv. Drug
Mumper, R.J.; Allen, D.D. J. Control. Release, 2003, 93, 271. Deliv. Rev., 2002, 54, 135.
[228] Koziara, J.M.; Lockman, P.R.; Allen, D.D.; Mumper, R.J. Pharm. [240] Vinogradov, S.V.; Batrakova, E.V.; Kabanov, A.V. Bioconjug.
Res., 2003, 20, 1772. Chem., 2004, 15, 50.
[229] Costantino, L.; Gandolfi, F.; Tosi, G.; Rivasi, F.; Vandelli, M.A.; [241] Kabanov, A.V.; Batrakova, E.V. Curr. Pharm. Des., 2004, 10,
Forni, F. J. Control. Release, 2005, 108, 84. 1355.
[230] Muller, R.H.; Mader, K.; Gohla, S. Eur. J. Pharm. Biopharm., [242] Lukyanov, A.N.; Torchilin, V.P. Adv. Drug Deliv. Rev., 2004, 56,
2000, 50, 161. 1273.
[231] Mehnert, W.; Mäder, K. Adv. Drug Deliv. Rev., 2001, 47, 165. [243] Kabanov, A.V.; Batrakova, E.V.; Melik-Nubarov, N.S.; Fedoseev,
[232] Wissing, S.A.; Kayser, O.; Muller, R.H. Adv. Drug Deliv. Rev., N.A.; Dorodnich, T.Y.; Alakhov, V.Y.; Chekhonin, V.P.;
2004, 56, 1257. Nazarova, I.R.; Kabanov, V.A. J. Control. Release, 1992, 22, 141.
[233] Yang, S.C.; Lu, L.F.; Cai, Y.; Zhu, J.B.; Liang, B.W.; Yang, C.Z. J. [244] Batrakova, E.V.; Li, S.; Vinogradov, S.V.; Alakhov, V.Y.; Miller,
Control. Release, 1999, 59, 299. D.W.; Kabanov, A.V. J. Pharmacol. Exp. Ther., 2001, 299, 483.
[234] Fundarò, A.; Cavalli, R.; Bargoni, A.; Vighetto, D.; Zara, G.P.; [245] Witt, K.A.; Huber, J.D.; Egleton, R.D.; Davis, T.P. J. Pharmacol.
Gasco, M.R. Pharm. Res., 2000, 42, 337. Exp. Ther., 2002, 303, 760.
[235] Wang, J.-X.; Sun, X.; Zhang, Z.-R. Eur. J. Pharm. Biopharm., [246] Muller, R.H.; Keck, C.M. J. Biotech., 2004, 113, 151.
2002, 54, 285. [247] Schöler, N.; Krause, K.; Kayser, O.; Muller, R.H.; Borner, K.;
[236] Göppert, T.M.; Müller, R.H. J. Drug Target, 2003, 11, 225. Hahn, H.; Liesenfeld, O. Antimicrob. Agents Chemother., 2001,
[237] Olbrich, C.; Gessner, A.; Kayser, O.; Müller, R.H. J. Drug Target, 45, 1771.
2002, 10, 387.
[238] Gessner, A.; Olbrich, C.; Schroder, W.; Kayser, O.; Müller, R.H.
Int. J. Pharm., 2001, 214, 87.

Received: December 07, 2005 Revised: March 15, 2006 Accepted: March 16, 2006