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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5443_Библиотеки_им_академика_М_И_Перельмана
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and drawbacks, with an emphasis on those that are most effective for screening potential
CNS medicines in a moderate- to high-throughput setting [7].
5.3.1 In vitro methods
5.3.1.1 Isolated brain capillaries
It is possible to isolate brain capillaries from several animal sources; however, these
are not the best for permeability screening since they cannot reach the luminal sur-
face of the isolated microvessels. As a result, drug transport from the blood into the
brain cannot be monitored; instead, only drug loss from the abluminal (brain) com-
partment can be measured. Given that it serves as the boundary between the blood
and the brain, the luminal surface is essential for permeability studies. It becomes dif-
ficult to precisely measure and evaluate drug permeability across the BBB without ac-
cess to the luminal surface. To solve this problem, new approaches or models must be
developed that allow for more direct access to the luminal surface of brain capillaries
for permeability screening [7].
5.3.1.2 Primary or low-passage brain capillary endothelial cell cultures
The in vivo BBB phenotype can be precisely simulated in primary or low passage
brain capillary endothelial cell cultures, offering important insights for understanding
drug transport. However, several components of the BBB, such as transporters and
enzymes, may be downregulated when endothelial cells are taken from the brain and
cultivated. For these cultures, capillary endothelial cells from bovine, pig, rat, or
human sources may be employed, while bovine and porcine cells are frequently cho-
sen because of their availability. Despite the possibility that some BBB-related proper-
ties have been downregulated in the grown cells, these cultures provide a useful
model for evaluating drug transport through the BBB [7, 24].
5.3.1.3 Immortalized brain endothelial cells
Several immortalized cell lines have been developed for the BBB permeability studies
to get around the problems with primary cell cultures. But the majority of these cell
lines, which are generat ed from rats, have a major drawback: they produce mono-
layers but fail to completely form tight junctions, creating a “leaky” barrier. Research-
ers have created cell lines that have been immortalized, transformed, transfected,
andtransducedtosolvethisproblem.TheRBE4cellline,RBEC1cellline,andTR-
BBB13 cell lines are a few examples of these cell lines. These cell lines were created by
5 Computational approaches to the prediction of the blood–brain distribution 93
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transfecting primary rat endothelial cells. The resultant cell lines show TEER values
that are reasonably low even after these modifications. These cell lines would there-
fore be better suited for other purposes for the assessment of endothelial cell uptake
of compounds rather than BBB permeability assessment [7, 25, 26].
5.3.1.4 Cells of non-cerebral origin
To study BBB permeability, researchers have looked into non-cerebral peripheral epi-
thelial cell lines as an alternative to immortalized brain endothelial cell lines. To eval-
uate P-glycoprotein’s (P-gp) role in BBB permeability, the multidrug resistance gene
(MDR1) was transfected into the Madin-Darby canine kidney (MDCK) cell line. Com-
pared to previous in vitro models, studies have revealed that MDR1-transfected MDCK
cells offer a better representation of in vivo BBB permeability. Though physically sim-
ilar to brain endothelial cells, MDCK cells have unique transport, metabolic, and
growth features. The MDCK cell line’s larger transverse area of intercellular connec-
tions causes it to overestimate paracellular transport, which is absent in BBB perme-
ability [7, 27, 28].
Table 5.1: Comparison of in vitro, in vivo, and in silico methods.
Method Definition Advantages Disadvantages
In vitro Studies conducted in
controlled laboratory
outside a living
organism.
Allows precise control over
experimental conditions.
Provides a simplified and cost-
effective model. Enables detailed
examination of specific
mechanisms. Reduces ethical
concerns associated with animal
testing.
Results may not fully represent
complex biological systems.
Limited physiological relevance.
Cannot capture interactions
with other organs/tissues in a
living organism.
In vivo Studies conducted
within a living
organism.
Reflects complex interactions
within a whole organism. Allows
assessment of systemic effects
and metabolism. Provides
relevant pharmacokinetic and
pharmacodynamic data.
Expensive, time-consuming, and
labor-intensive. Ethical
considerations and regulatory
limitations associated with
animal experimentation.
Variability among individual
organisms.
In silico Studies conducted
using computer
simulations.
Enables rapid screening of large
chemical libraries. Cost-effective
compared to experimental
approaches. Facilitates
predictions of drug–target
interactions.
Relies heavily on accuracy of
input data. Limited accuracy in
capturing the complexity of
biological systems. Uncertainty
in simulating real-world
scenarios.
94 Sarwal Amita, Bharti Sunil, and T.V.S. Padmajyoti
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5.3.1.5 Immobilized artificial membranes
For evaluating drug permeability through cell membranes, immobilized artificial
membranes (IAMs) have been offered as a substitute model. Phosphatidylcholine resi-
dues bonded to silica propylamine form IAMs, which resemble fluid membranes and
resemble a lipid bilayer. They are employed in high-performance liquid chromatogra-
phy as a chromatographic interface. IAM retention has been investigated as a poten-
tial predictor of brain penetration, especially for substances that passively cross the
BBB. IAM retention factors and the uptake of different drugs by the brain, including
basic, neutral, and acidic substances, have shown a correlation in some studies. The
regression can be improved by taking into account variables like ionization and solute
size. IAMs have low predictive value when brain uptake is modified by plasma pro-
tein binding, active transport, active efflux, or metabolism and do not recreate the
process of diffusion across a membrane. IAMs have several limitations when it comes
to simulating sophisticat ed transport mechanisms across the BBB, even if they may
offer insights into how solutes are partitioned into membranes [7, 29–31].
5.3.2 In vivo models
Despite the positive aspects of in vitro models and their suitability for moderate- to
high-throughput screening, it is vital to not rely only on these techniques for BBB re-
search. Correlating in vitro and in vivo investigations will help create a thorough under-
standing. The single carotid injection method, in situ perfusion method, intravenous
injection method, brain efflux index method, and intracerebral microdialysis method
have all been used to evaluate drug uptake into the brain. With the help of these techni-
ques, it is possible to calculate the BBB permeability-surface area (PS) product, or
the logBB, which measures a substance’s brain-to-blood ratio at a specific time. The uni-
directional clearance of blood through the BBB, measured in mL min
−1
g
−1
, is repre-
sented by the BBB PS product [7, 8, 32].
5.3.2.1 Carotid artery single injection technique
The brain uptake index (BUI) or carotid artery single injection technique is a method
for measuring a compound’s brain uptake. The carotid artery is cannulated, followed
by a quick single injection of the chemical along with a radiolabeled diffusible refer-
ence compound as an internal standard. The internal standard’s goal is to determine
the amount of injected substance that is distributed to the brain. Following injection,
the drug quickly travels through the brain, and the animal is decapitated. The BUI is
then calculated by analyzing the brain and injection solution. The technique pre-
sumes that the reference compound readily diffuses across the BBB and that there is
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no drug back-diffusion from the brain to the blood. However, due to the short capil-
lary transit time, measurements of BBB PS products are limited to less than 10 mL
min
−1
g
−1
. Furthermore, because the exterior arteries are not occluded, the drug may
spread throughout the body, with just a small percentage (about 10%) reaching the
brain [7].
5.3.2.2 In situ perfusion technique
The in situ perfusion technique developed by Takasato et al. (1984), is an extension of
the carotid artery single injection technique, which is used to monitor drug uptake into
the brain. In this approach, the animal is given anesthesia, and a perfusion catheter is
inserted into the external carotid artery. The perfusion fluid is then administered retro-
gradely down the external carotid artery and up the internal carotid artery towards the
brain. To prevent perfusion fluid from mingling with systemic circulation, the common
carotid artery is occluded. After perfusion, the animal is decapitated and the drug con-
centration is evaluated to calculate the BBB PS product. A reference chemical, in addi-
tion to the compound of interest, is supplied to quantify brain plasma volume. A
capillary depletion technique can be utilized to differentiate between endothelial bind-
ing/endothelial endocytosis and transcytosis. Alternatively, a physiological buffer post-
perfusion wash might be used. The advantages of in situ perfusion include the avoid-
ance of systemic exposure and the ability to adjust the perfusion fluid concentration.
The technique, however, necessitates the use of numerous animals and a large amount
of analytical time, making it unsuitable for high-throughput screening [7, 33].
5.3.2.3 Intravenous injection technique
The intravenous injection approach is regarded as the “gold standard” for determin-
ing the permeability of the BBB. To give the test drug, a femoral vein is cannulated, or
a tail vein injection is performed in rats or mice. At several intervals during the ex-
periment, arterial blood samples are obtained to determine the drug levels in the
plasma and brain. For adjusting drug distribut ion in the brain microvasculature, a
plasma volume marker is also given. The capillary depletion technique can be used to
distinguish bound and transcytosed drug molecules. The potential advantages of this
approach include t he calculation of direct pharmacokinetic parameters, improved
sensitivity, and preservation of the BBB’s physiological conditions. However, one sig-
nificant disadvantage is the possibility of substantial metabolism and diffusion into
peripheral organs, which can lead to inaccurate outcomes. Back-diffusion from the
brain to plasma is also possible at later time points. Despite these drawbacks, the in-
travenous approach gives a realistic assessment of brain levels that closely reflects
the human scenario [7, 34, 35].
96 Sarwal Amita, Bharti Sunil, and T.V.S. Padmajyoti
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5.3.2.4 Intracerebral microdialysis
Intracerebral microdialysis is a procedure that uses a dialysis fiber implanted into the
brain to directly sample brain interstitial fluid. It allows for the monitoring of drug con-
centrations in the brain following oral, intravenous, or subcutaneous delivery. Small
compounds can diffuse from higher to lower concentrations via a semipermeable mem-
brane when the microdialysis probe is perfused with a physiological solution. The main
advantage of this technique is that it provides pharmacokinetic profiles of substances
in the brain without requiring several animals to be sacrificed at different time inter-
vals. It also enables the determination of brain inflow and efflux kinetics. The probe
can be inserted in certain brain regions, allowing compounds to be targeted to specific
places. As small amounts of substances may be present in the dialysate, the procedure
relies on the sensitivity of the assay method. Another issue is that the probe’s implanta-
tion can disrupt the BBB, potentially leading to chronic BBB disruption [7, 36]. Figure 5.2
Enlists various approaches for brain targeted drug delivery.
NOVEL APPROACHES
BIOTECHNOLOGY BASED
APPROACHES
CHEMISTRY BASED
APPROACHES
APPROACHES FOR
BRAIN TARGETED
DRUG DELIVERY
DIERECT CNS DELIVERY
DIRECT
DELIVERYSYSTEMIC
Figure 5.2: Approaches for brain targeted drug delivery.
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5.4 Various approaches for brain-targeted
drug delivery
5.4.1 Direct systemic delivery
5.4.1.1 Intravenous delivery
This approach is commonly used to inject drugs directly into the circulation, poten-
tially enabling delivery to the brain. Drug delivery through this route is facilitated by
the large surface area of the brain capillary network. As the neurons in the brain are
well connected with the blood vessels, the delivered drug can get access to the brain
by crossing the vascular barrier, enabling drugs to reach almost all areas of the brain.
The availability of drugs in the brain following IV injection is influenced by factors
such as drug plasma half-life, metabolism, drug-plasma protein binding, BBB perme-
ability, and transport into peripheral organs [37–39]. When given by this route, sev-
eral drugs have shown a therapeutic effect in clinical trials. Slow IV treatment of
neurotrophin, in a study, led to a considerable decrease in cortical stroke volume in
stroke patients [40]. When rats with intracranially transplanted glioblastomas were
administered Doxorubicin intravenously using polysorbate 80-coated nanoparticles, a
40% cure was exhibited [37, 40, 41].
– Concentration gradient of drug
– Molecular weight of the drug
– Lipophilicity of drug
– Affinity for efflux proteins
FACTORS
AFFECTING
DRUG
TRANSPORT
ACROSS BBB
– Sequestration by other cells
– Clearance rate of the drug
– Pathological status
– Cellular enzymatic stability
– Systemic enzymatic stability
Figure 5.3: Factors affecting drug transport across BBB.
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5.4.1.2 Intra-arterial delivery
The intra-arterial approach, which involves injecting drugs into the circulation, is an
alternative to intravenous administration for delivering drugs directly to the brain.
This route allows drugs to access the brain vasculature before reaching peripheral tis-
sues, thereby avoiding first-pass metabolism. It is frequently used for localized delivery
of drugs to brain tumors. The bioavailability of the drug in the brain is determined by
its passage via capillaries, reaching the choroid plexus epithelium, or entering the CSF
via the white matter and perivascular pathways. In a human malignant glioma model,
better anticancer results were achieved through intra-arterial administration of a p-53
(tumor suppressor gene) containing adenoviral vector [37, 42].
5.4.1.3 Intranasal delivery
The intranasal route delivers drugs to the brain in a way that is not invasive. It offers
better targeting capabilities as the drug directly moves from the nasal submucosa to
the CSF compartment of the brain. The olfactory region, adjacent to the respiratory
region, is a key site where drugs can be absorbed directly into the brain through vari-
ous mechanisms, including transcellular and paracellul ar routes, as well as via tri-
geminal neural pathway, bypassing the BBB and achieving high concentrations in the
CSF. The olfactory region of nasal mucosa contains olfactory cells that extend up into
the cranial cavity. When the drug formulation on nasal installation comes in contact
with the mucosa it is rapidly transported directly into the brain, skipping the BBB,
and achieving very rapid CSF levels [37, 43].
Intranasal administration has several advantages, including reduced dosage, self-
administration, and prevention of the use of surgical methods. But there are several
drawbacks, such as potential nasal mucosa injury, rapid mucociliary clearance of the
drug, interference owing to nasal congestion, and nasal mucosa irritations. The intranasal
route has been proven in recent research to be successful for a variety of drugs and pep-
tides. In a rat model of ischemia, in a recent study, NAD+ delivered intranasally decreased
brain damage. Gallotannin, a poly (ADP-ribose) glycohydrolase (PARG) inhibitor, when
administered intranasally, reduced the occurrence of ischemic brain damage [37, 44, 45].
5.4.2 Direct CNS delivery
5.4.2.1 Intracerebral delivery
Intracerebral (intraparenchymal) delivery is a method that delivers the drugs directly
into the brain parenchyma. Drugs can be administered through intrathecal catheters
via bolus injection or continuous infusion. Bolus injection, however, faces challenges
5 Computational approaches to the prediction of the blood–brain distribution 99
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due to the limited diffusion coefficient caused by the densely packed arrangement of
cells in the brain and concentration-dependent diffusion phenomena. To overcome
these challenges, continuous infusion methods, such as convection-enhanced diffu-
sion (CED), have been developed. CED has shown superior ability to deliver drugs in
large doses, maintaining drug concentration and distribution over time. Compared to
bolus injection, CED demonstrated significantly higher drug concentrations in the pa-
renchyma. Immunotoxins and drugs like paclitaxel have been successfully delivered
using CED-mediated drug delivery [37, 46, 47].
Intracerebral implants are devices that use biodegradable or nonbiodegradable
polymers to deliver drugs to specific sites in the brain in a controlled release manner.
An implant containing nerve growth factor placed in the brain of a quadriplegic pa-
tient for treatment showed better results from spinal cord damage. Recently, an os-
motic pump-type device, Ommaya reservoir, which has a refillable reservoir and
catheter, was implanted into the lateral ventricle and it had shown better cerebral
concentration [37, 48].
5.4.2.2 Intraventricular delivery
Intraventricular delivery, also known as transcranial drug delivery, involves the di-
rect administration of therapeutic agents into the cerebral ventricles. This route is
beneficial for bypassing the BBB and distributing drugs mainly into the ventricles and
subarachnoid areas of the brain. Intraventricular delivery is particularly suitable for
treating meningiomas and metastatic cells in the CSF. The advantage of this route is
the achievement of higher drug concentrations in the brain compared to extravascu-
lar distributions due to the lack of interconnecti on with an interstitial fluid of the
brain, unlike intra cerebral delivery. However, potential d isadvantages include the
risk of causing subependymal astrogliatic reactions [37, 49].
5.4.2.3 Intrathecal delivery
Intrathecal drug administration is a less invasive way of directly delivering drugs into
the brain’s cisterna magna. But, it fails to result in drug accumulation in the deep
brain parenchyma. Also drug delivery by this route can cause drug spreading down
the spinal canal, which is a major disadvantage and it was observed when etoposide,
after being given through this route to dogs, led to ataxia and loss of muscle coordina-
tion [37, 49].
100 Sarwal Amita, Bharti Sunil, and T.V.S. Padmajyoti
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5.4.2.4 BBB disruption
BBB disruption is a method of delivering drugs directly to the CNS by disrupting the
BBB’s tight junctions. This can be accomplished by the use of chemical compounds or
the use of external energy. One approach is to use hyperosmolar chemicals, such as
hypertonic solutions, to enhance osmotic pressure and open tight junctions. Mannitol
is the most common osmotic substance, which is used in conjunction with the drug
and is supplied by intracarotid arterial infusion. This leads to the shrinking of endo-
thelial cells, disarrangement of extracellular proteins, and drug entry through the
paracellular route. Bradykinin and its analogs can also alter tight junctions on endo-
thelial cells via modulation of B2 receptors, enhancing BBB permeability. Another
method of disrupting the BBB is to use ultrasound and electromagnetic radiation to
target specific sites of the brain. There are three hypothesized processes for this
method: heat effects, cavitation effects, an d microbubble generation. Through ultra-
sonic frequencies, thermal effects cause altered permeability and generalize d BBB
opening. The cavitation effect causes air-filled spaces in the barrier, allowing drugs to
get through. An ultrasonic contrast agent causes microbubble production, which in-
creases in size with time and bursts finally, opening tight junctions without causing
tissue injury. Noninvasive ultrasound-mediated BBB disruption is extensively em-
ployed in diagnostics and brain imaging [37]. Factors affecting drug transport across
BBB have been shown in Fig 5.4.
5.4.3 Chemistry-based approaches
Significant research has been conducted in recent years to identify effective strategies
for delivering neurotherapeutics across the BBB. The utilization of chimeric peptides
and cationic proteins are two techniques that have received a lot of attention.
5.4.3.1 Chimeric peptides
To construct a molecule capable of effective transport, chimeric peptides combine a
drug that cannot cross the BBB with a transport vector. An endogenous peptide,
monoclonal antibody (mAb), modified protein, or peptidomimetic antibody can be
used as the vector. To enhance brain distribution, these peptides use a variety of
transport mechanisms, including peptide-specific receptors. Insulin and transferrin
are two examples of circulating peptides that cross the BBB via their respective BBB
receptors. The interaction of the peptide vector with its binding receptor is critical for
brain targeting. Endogenous peptides such as cationized albumin, mAbs, and histones
have been studied as possible brain drug delivery vectors [37, 50, 51].
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5.4.3.2 Cationic proteins
Cationic proteins provide a method for delivering proteins and peptides with basic
isoelectric points to the brain. Proteins have a large molecular weight that hinders
their ability to cross the BBB. Modifying them into their cationic form increases their
net positive charge, allowing them to interact electrostatically with anionic functional
groups present on the brain surface and thus enabling them to enter the brain [37].
5.4.3.3 Prodrugs
In 1986, Garzon-Aburbeh et al. proposed that prodrugs (which are inactive, modified
derivatives of active pharmaceutical ingredients (APIs)) are helpful in the delivery of
hydrophilic drugs to the brain. After reaching the brain via the circulation, these pro-
drugs are metabolized by the enzymes of BBB-producing active forms of drugs, which
can cross the BBB and achieve therapeutic concentrations. For delivering drugs to the
brain, prodrugs provide increased site specificity and lower toxicity. Examples include
prodrugs of
L-dopa for treating Parkinsonism and prodrugs of antiviral agents such as
Zidovudine for the treatment of viral infections [37, 52, 53].
5.4.4 Novel approaches
Novel drug delivery has evolved as an effective approach for delivering drugs to the
brain. Small colloidal particles are used as carriers in this strategy. Due to their con-
trolled drug-release properties and ability to precisely target particular areas, these
particles are preferred.
5.4.4.1 Liposomes
Liposomes are biocompatible and biodegradable lipid-based carriers composed of phos-
pholipids and sphingolipids. They offer the advantage of encapsulating hydrophilic, li-
pophilic, and amphoteric drug molecules either inside the liposome or on its surface.
Liposomal technology has advanced significantly, allowing for the design of carriers
with improved site-specific targeting. To target the brain, long-circulating liposomes can
be modified with specific targeting vectors such as mAbs or cationized proteins. These
liposomes can cross the BBB through absorptive-mediated or receptor-mediated trans-
cytosis. Cationic liposomes have demonstrated effective BBB penetration through ab-
sorptive-mediated transcytosis, while liposomes with mannose coating are commonly
used for brain targeting. Sterically stabilized liposomes, achieved through surface modi-
fication with certain chemical substances, have emerged as a promising technology for
102 Sarwal Amita, Bharti Sunil, and T.V.S. Padmajyoti
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