brain targetting

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Drug Targeting
Dr. Suresh Bandari
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The main complications currently associated with
systemic drug administration are

• Even biodistribution of pharmaceuticals
  throughout the body
• The lack of drug specific affinity toward a
  pathological site
• The necessity of a large total dose of a drug to
  achieve high local concentration
• Non-specific toxicity and other adverse
  side-effects.

Drug targeting may resolve many of these problems
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Drug targeting is the ability of the drug to
accumulate in the target organ or tissue
selectively and quantitatively, independent of
the site and methods of its administration.
Drug administration protocols may be simplified
Drug quantity may be greatly reduced as well as
the cost of therapy Drug concentration in the
required sites can be sharply increased without
negative effects on non-target compartments.
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MAGIC BULLET CONCEPT OF PAUL EHRLICH

• Drugs would be targeted by virtue of groups
  having affinity
• for specific cells
• A ligand would confer specificity on a
  non-specific reagent

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• MAGIC BULLET Two components
• The first one is recognizes and binds the target
• The second one provides a therapeutic action in
  this
• target

Currently, the concept of magic bullet includes a
coordinated behavior of three components (a)
drug (b) targeting moiety (c)
pharmaceutical carrier
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• The principal schemes of drug targeting include
• Direct application of a drug into the affected
  zone,
• Passive drug targeting (spontaneous drug
  accumulation in the areas with leaky vasculature,
  or Enhanced Permeability and Retention-EPR-effect)
  ,
• Physical targeting (based on abnormal pH value
  and/or temperature in the pathological zone),
• Magnetic targeting (or targeting of a drug
  immobilized on paramagnetic materials under the
  action of an external magnetic field), and
• Targeting using a specific vector molecules
  (ligands having an increased affinity toward the
  area of interest).

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a) Macro molecular conjugates, b) Particulate
drug carriers
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• Pharmaceutical carriers
• polymers
• microcapsules
• microparticles
• Nanoparticles
• lipoproteins
• liposomes
• micelles

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Targeting Moieties

• Antibodies
• Lectins and other proteins
• Lipoproteins
• Hormones
• Charged molecules
• Polysaccharides
• Low-molecular-weight ligands

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Brain Targeting
Delivery of drugs to the brain is a major
challenge because it is tightly segregated from
the circulating blood by a unique membranous
barrier, the bloodbrain barrier (BBB). The
brain and spinal cord are lined with a layer of
special endothelial cells that lack fenestrations
and are sealed with tight junctions that greatly
restrict passage of substances from the
bloodstream than endothelial cells in capillaries
elsewhere in the body. These endothelial cells,
together with perivascular elements such as
astrocytes and pericytes, constitute the BBB.
BBB is often the rate-limiting factor in
determining permeation of therapeutic drugs into
the brain.
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Characteristics of the BBB are indicated (1)
tight junctions that seal the pathway between the
capillary (endothelial) cells (2) the lipid
nature of the cell membranes of the capillary
wall which makes it a barrier to water-soluble
molecules (3), (4), and (5) represent some of
the carriers and ion channels (6) the 'enzymatic
barrier 'that removes molecules from the blood
(7) the efflux pumps which extrude fat-soluble
molecules that have crossed into the cells
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• The factors affecting particular substance to
  cross BBB
• Drug related factors at the BBB
• Concentration at the BBB and the size,
• Flexibility,
• Conformation,
• Ionization (nonionized form penetrates BBB)
• Lipophilicity of the drug molecule,
• Cellular enzyme stability and cellular
  sequestration,
• Affinity for efflux mechanisms (i.e.
  P-glycoprotein),
• Hydrogen bonding potential (i.e. charge),
• Affinity for carrier mechanisms, and
• Effect on all of the above by the existing
  pathological conditions

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• The physicochemical characteristics
• Log Po/w of the therapeutic agent, the rule of 2
  is generally accepted i.e. the value of log Po/w
  nearing 2 is considered optimal.
• However, increasing the lipophilicity with intent
  to increase permeability would increase the
  volume of distribution (Vd) and also the rate of
  oxidative metabolism by cytochrome P450
• Peripheral factors including systemic enzymatic
  stability,
• Plasma protein binding affinity,
• Uptake of the drug into other tissues,
• Clearance rate, and
• Effects of existing pathological conditions are
  also important.

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• The lipophilicity of a given drug is inversely
  related to the degree of hydrogen bond formation
  that occurs with surrounding water.
• The presence of certain chemical moieties in drug
  like terminal amide, primary amines or amides and
  hydroxyl group favors hydrogen bond formation
  resulting in a decreased lipophilicity.
• Thus for a compound to be transported through the
  BBB, the cumulative number of hydrogen bonds
  should not go beyond 810.
• Therefore for small drugs increasing
  lipophilicity i.e. decreasing hydrogen bonds has
  a positive impact on capillary permeability and
  drug transfer to the brain and for large drug
  molecules with molecular weight above 400 Da or
  for those with strong polarity, the capillary
  permeability will remain low regardless of the
  lipophilicity

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Several specialized transport mechanisms of
solute transfer across endothelial cells and into
the brain interstitium are also present within
the BBB Carrier system for monosaccharides,
monocarboxylic acid, neutral amino acids, basic
amino acid, acidic amino acids, amines, purine
bases, nucleosides, vitamins and hormones. The
more lipophilic substances that are present in
the blood can diffuse passively directly through
the lipid of the cell membrane and enter the
endothelial cells and brain by this means.
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These solutes, and in many cases their
metabolites, are actively removed from the CNS by
efflux transporters. Various efflux transport
pathways like P-glycoprotein and active organic
acid present in choroids plexus may also be
active in brain endothelial cells efflux systems
are present in the BBB to remove unwanted
substances, On the other hand the presence of the
tight junctions and the lack of aqueous pathways
between cells greatly restrict the movement of
polar solutes across the cerebral endothelium
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• The molecules that can freely diffuse through
  this capillary endothelial membrane can passively
  cross the BBB, and this ability is closely
  related to their lipid solubility (lipophilicity/
  hydrophobicity).
• Practically all drugs currently used to treat
  brain disorders are lipid-soluble and can readily
  cross the BBB following oral administration.
• The BBB also has an additional, enzymatic aspect
  solutes crossing the endothelial cell membrane
  are subsequently exposed to numerous degrading
  enzymes within these cells.

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• These cells also contain many mitochondria
  metabolically active organelles and active
  transport can significantly alter both inward and
  outward transport for compounds.
• The BBB is highly efficient and makes the brain
  practically inaccessible to lipid-insoluble
  compounds.
• Brain-delivery of such compounds, therefore,
  requires a strategy to overcome the BBB.
• Delivery of compounds such as neuropeptides or
  oligonucleotides is further complicated by their
  metabolic lability.

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• Functions of the BBB
• Firstly, maintaining internal environment of the
  brain, i.e. maintaining brain interstitial fluid
  (ISF) and the cerebrospinal fluid (CSF)
  composition within extremely fine limits, far
  more so than the somatic extracellular fluid, so
  that the neurones can perform their complex
  integrative functions.
• BBB protects the brain from fluctuations in ionic
  composition that can occur after a meal or
  exercise, which could disturb synaptic and axonal
  signaling.
• The barrier helps to keep the centrally and
  peripherally acting neurotransmitters separate.

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• A major function of the BBB is neuroprotection.
  Over a lifetime CNS will be exposed to a wide
  range of neurotoxic metabolites and acquired
  xenobiotics, which may cause cell damage and
  death. As neuronal replacement is virtually
  absent in the CNS of mammals, any enhancement of
  neuronal death will result in accelerating
  degenerative pathologies and advance natural
  debilitation with age.
• Finally the continual turnover and drainage of
  CSF and ISF by bulk flow helps to clear larger
  molecules and brain metabolites, thus maintaining
  brain microenvironment

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Strategies for Brain Targeting Mechanisms for
drug targeting in the brain involve going either
"through" or "behind" the BBB. Neurosurgical or
Invasive Strategies BBB disruption Disruption of
BBB by osmotic means (Hyperosmolar solutions),
Intraventricular drug infusion Intracerebral
Implants Biodegradable implants, Physiologic
based strategies Psuedo nutrients eg
L-dopa Cationic antibodies These undergo
Absorption mediated trancytosis through BBB owing
to positive charge. Chimeric peptides
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Pharmacologic Strategies Chemical Delivery
system Nanocarriers for active targeting of the
brain Liposomes Polymeric micelles. Polymeric
nanoparticles Lipid nanoparticles
. Biochemically by the use of vasoactive
substances such as bradykinin, Localized
exposure to high intensity focused ultrasound
(HIFU). Cell-penetrating peptides and Brain
transport vectors
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Chemical Delivery Systems
Brain-targeted chemical delivery systems (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. By localizing drugs at their desired
site of action, one can reduce toxicity and
increase treatment efficiency. The CDS concept
evolved from the prodrug concept in the early
1980s, but was differentiated by the introduction
of target or moieties and the use of multistep
activation. The cunning aspect of these
brain-targeted systems is that, in addition to
providing access by increasing the lipophilicity,
they exploit the specific bidirectional
properties of the BBB to lock inactive drug
precursors in the brain on arrival, preventing
exit back across the BBB
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CDSs are inactive chemical derivatives of a drug,
being obtained by one or more chemical
modifications. The introduced bioremovable
moieties can be categorised into two types. A
targetor (T) moiety is responsible for targeting,
site-specificity and lock-in whereas modifier
functions (F1...Fn) serve as lipophilizers,
protect certain functions, i.e., necessary
molecular properties to prevent premature,
unwanted, metabolic conversions. The CDS is
designed to undergo sequential metabolic
conversions, disengaging the modifier function(s)
and finally the targetor, after the moiety has
fulfilled its site- or organ-targeting role
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Lock in mechanism of E2-CDS provided by
introduction of a targetor moiety that exploits a
1,4-dihydrotrigonelline (green) Trigonelline
(red) type conversion. On hydrolysis trigonelline
converts to active drug.
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During the past decade, the system has been
explored with a wide variety of drug classes, and
considerably increased brain exposure as well as
brain targeting (i.e. brain vs systemic exposure)
have been obtained in several cases for example,
3-azido-3-deoxythymidine (AZT)-CDS,
ganciclovir-CDS and benzylpenicillin-CDS. AZT-CDS
administration in rats simultaneously increases
brain exposure 32-fold and decreases blood
exposure threefold as compared with AZT
administration. Among all CDSs, the estradiol
chemical delivery system (E2-CDS) is in the most
advanced investigation stage. Following earlier
clinical trials (Phase I and II),
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• Molecular packaging brain delivery of
  Neuropeptides
• Delivery of peptides through the BBB is even more
  challenging than delivery of other drugs, because
  peptides tend to be rapidly inactivated by the
  ubiquitous peptidases.
• For a successful delivery, three issues have to
  be solved simultaneously
• enhance passive transport by increasing the
  lipophilicity,
• ensure enzymatic stability to prevent premature
  degradation, and
• exploit the lock-in mechanism to provide
  targeting.
• Successful brain deliveries have already been
  achieved using this strategy for a Leu-enkephalin
  analog, thyrotropin-releasing hormone (TRH)
  analogs and kyotorphin analogs

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It is of particular significance for TRH
delivery because the corresponding process might
require up to five or six consecutive metabolic
steps. Therefore, selection of a suitable spacer
moiety, which is inserted between the targetor
and peptide units to ensure correct timing for
targetor release, proved important for the
efficacy of TRH-CDSs.
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• Dopamine is also classed as a monoamine
  neurotransmitter and is concentrated in very
  specific groups of neurons collectively called
  the basal ganglia. Dopaminergic neurons are
  widely distributed throughout the brain in three
  important dopamine systems (pathways) the
  nigrostriatal, mesocorticolimbic, and
  tuberohypophyseal pathways. A decreased brain
  dopamine concentration is a contributing factor
  in Parkinson?s disease, while an increase in
  dopamine concentration has a role in the
  development of schizophrenia.

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The first group regulates movements a deficit of
dopamine in this (nigrostriatal) system causes
Parkinson's disease which is characterized by
trembling, stiffness and other motor disorders,
while in the later phases dementia can also set
in. ?The second group, the mesolimbic, has a
function in regulating emotional behavior. The
third group, the mesocortical, is involved with
various cognitive functions, memory, behavioral
planning and abstract thinking, as well as in
emotional aspects, especially in relation to
stress. The earlier mentioned reward system is
part of this last system. ?Disorders in the
latter two systems are associated with
schizophrenia.
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In Parkinsons disease, there is degeneration of
the substantia nigra which produces the chemical
dopamine deep inside the brain
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Since PD is related to a deficiency of dopamine,
it would be appropriate to administer
dopamine Problem Dopamine does not cross BBB,
since it is too polar
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If dopamine is too polar to cross the BBB, how
can L-DOPA cross it?
L-DOPA is transported across the BBB by an amino
acid transport system (same one used for tyrosine
and phenylalanine)
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Once across, L-DOPA is decarboxylated to dopamine
by Dopa Decarboxylase.This is an example of a
prodrug, that is, a molecule that is a
precursor to the drug and is converted to the
actual drug at an appropriate place in the body.
In actual practice, L-DOPA is almost always
coadminstered together with an inhibitor of
aromatic L-amino acid decarboxylase, so it
doesnt get converted to dopamine before it
crosses the BBB. The inhibitor commonly used is
carbidopa, which does not cross the BBB itself.
The inhibitor also prevents undesirable side
effects of dopamine release into the PNS,
including nausea.
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• Polymeric nanoparticles suitable delivery systems
  for brain.
• The mechanisms for nanoparticle mediated drug
  uptake by the brain include
• Enhanced retention in the brainblood
  capillaries, with an adsorption on to
• the capillary walls, resulting in a high
  concentration gradient across the BBB.
• Opening of tight junctions due to the presence
  of nanoparticles.
• Transcytosis of nanoparticles through the
  endothelium.
• Furthermore, coating of these polymeric
  nanoparticles with polysorbate has been reported
  to improve the brain bioavailability. Some of the
  proposed mechanisms by which the polysorbate
  coating is effective, include
• Solubilization of endothelial cell membrane
  lipids and membrane
• fluidization, due to surfactant effects of
  polysorbates.
• Endocytosis of polymeric nanoparticles due to
  facilitated interaction with
• BBB endothelial cells.
• Inhibition of efflux system, especially P-gp.

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• But, there are various problems associated with
  the use of these polymeric nanoparticles
• Residual contamination from the production
  process, for example by
• organic solvents,
• Polymerization initiation,
• Large polymer aggregates,
• Toxic monomers and toxic degradation products,
• Expensive production methods,
• Lack of large scale production method and
• A suitable sterilization method e.g.
  autoclaving.
• Considering the success of nanoparticles to
  pass through the BBB and their limitation(s)
  especially toxicity and stability, another
  suitable option for drug delivery into the brain
  would be SLNs.

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SOLID LIPID NANOPARTICLES SLNs constitute an
attractive colloidal drug carrier system. SLNs
consist of spherical solid lipid particles in the
nanometer range, which are dispersed in water or
in aqueous surfactant solution. They are
generally made up of solid hydrophobic core
having a monolayer of phospholipid
coating. Advantages of SLNs over polymeric
nanoparticles (and other delivery systems like
liposomes) The nanoparticles and the SLNs
particularly those in the range of 120200 nm are
not taken up readily by the cells of the RES
(Reticulo Endothelial System) and thus bypass
liver and spleen filtration. 2. Controlled
release of the incorporated drug can be achieved
for upto several weeks. Further, by coating with
or attaching ligands to SLNs, there is an
increased scope of drug targeting.
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3. SLN formulations stable for even three years
have been developed. 4. High drug
payload. 5.Excellent reproducibility with a cost
effective high pressure homogenization method as
the preparation procedure. 6.The feasibility of
incorporating both hydrophilic and hydrophobic
drugs. 7. The carrier lipids are biodegradable
and hence safe. 8. Avoidance of organic
solvents. 9. Feasible large scale production and
sterilization.
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Use of ligands. Ligands or homing devices that
specifically bind to surface epitopes or
receptors on the target sites, can be coupled to
the surface of the long-circulating
carriers. Certain cancer cells over express
certain receptors, like folic acid
(over-expressed in cells of cancers with
epithelial origin), LDL (B16 melanoma cell line
shows higher expression of LDL receptors) and
peptide receptors (such as somatostatin analogs,
vasoactive intestinal peptide, gastrin related
peptides, cholecystokinin, leutanising hormone
releasing hormone). Attaching suitable ligands
for these particular receptors on to the
nanoparticles would result in their increased
selectivity Allen et al. postulated that the
presence of specific ligands on the surface of
nanoparticles could lead to their increased
retention at the BBB and a consequent increase in
nanoparticle concentration at the surface of BBB.
While attempting to prove their assumption, they
prepared coated nanoparticles from Brij 78, and
emulsifying wax, with thiamine ligand (linked to
DSPE via a PEG spacer).
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Gene targeting technology gene therapy of the
brain
Many serious disorders of the CNS that are
resistant to conventional small-molecule therapy
could be treated, even cured, with gene therapy
of the brain. Current approach include delivery
of the therapeutic gene to the brain by drilling
a hole in the head followed by insertion of the
gene incorporated in a viral vector. The
advantage of craniotomy-based gene delivery is
that the gene can be expressed in a highly
circumscribed area of the brain with an effective
treatment volume of 110 µl. This craniotomy
based delivery does not enable the expression of
the therapeutic gene widely throughout the brain
or even to a relatively localized area such as a
brain tumor, which could have a volume greater
than several milliliters. Viruses have been the
vector of choice because the virus-coat proteins
trigger endocytosis of the virus into the target
brain cell. The two most commonly used viral
vectors are adenovirus or herpes simplex virus
(HSV). The problem with both these viruses is
that, because they are common, humans have a
pre-existing immunity. This immunity generates an
inflammatory response
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Gene targeting technology Craniotomy and viruses
are first-generation brain gene delivery systems.
Gene therapy of the brain use delivery systems
that are both noninvasive and non-viral. A brain
gene delivery system should enable widespread
expression of a therapeutic gene throughout the
brain following a simple intravenous injection.
First, the exogenous gene packaged in a
non-viral plasmid vector is interiorized within a
nanocarrier, much like exogenous genes are
packaged in the interior of viruses. This
protects the therapeutic gene from the
endonucleases in the body. Second, the
nanocarrier is non-immunogenic and formed by
either natural lipids or other non-immunogenic
polymeric substances. Third, the nanocarrier
carrying the exogenous gene is stable in the
bloodstream with optimal plasma pharmacokinetics
following an intravenous injection. (The rapid
RES uptake can be blocked by pegylation. The
pegylated liposomes are stable in the bloodstream
and have long blood circulation times).
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Fourth, the surface of the nanocarrier is
modified that triggers transcytosis across
microvascular endothelial barriers such as the
BBB and then endocytosis into target neurons or
glial cells in brain. (Targeting through the BBB
and neuronal plasma membrane is accomplished by
tethering the tips of 12 of the PEG strands
with a targeting monoclonal antibody (MAb) to
form an immunoliposome). Owing to expression of
the transferrin receptor (TfR) on both the BBB
and the neuronal plasma membrane, the use of an
anti-TfR MAb causes the pegylated immunoliposome
to undergo transport through both the BBB and the
neuronal plasma membrane in vivo. The liposomal
lipids fuse with the endosomal membrane inside
neurons, which releases the plasmid into the
cytosolic space of target neurons, where it can
then diffuse to the nuclear compartment. The only
immunogenic component of the formulation is the
MAb and the immunogenecity of murine MAbs in
humans can be eliminated with genetic engineering
and humanization of the MAb.
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ß-Galactosidase histochemistry of a rat brain
removed 48 h after a single intravenous injection
of a ß-galactosidase gene carried by a plasmid
that is packaged in the interior of 85 mm
liposomes. The surface of the liposome is
covered by thousands of strands of 2000 Da (PEG),
and this stabilizes the liposome in the blood and
prolongs the circulation time in the plasma.
Approximately 2 of the PEG strands that project
from the liposome surface are tethered to a
monoclonal antibody that targets the transferrin
receptor. This receptor is expressed both on the
brain capillary endothelium, which forms the
bloodbrain barrier in vivo, and on the neuronal
plasma membrane. Targeting the immunoliposomes to
the transferrin receptor enables transport across
both the bloodbrain barrier and the neuronal
plasma membrane in vivo. The use of gene
targeting technology enables widespread
expression in the brain of an exogenous gene
following a single intravenous administration of
a non-viral gene formulation.
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• Nice presentation
• All the credit goes to Dr. Suresh Bandari
• Thank you.

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