The Blood-Brain Barrier and CNS Drug Development Dr ...and CNS Drug Development Dr....
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The Blood-Brain Barrier
and CNS Drug Development
Dr. Danica Stanimirovic
The screen versions of these slides have full details of copyright and acknowledgements 1
The Blood-Brain Barrier and CNS Drug Development
Dr. Danica Stanimirovic
Director, Neurobiology Program
National Research Council of Canada
Institute for Biological Sciences
"The Program is committed to understanding the molecular nature
of neurodegenerative diseases… Our hope is that our discoveries will one day
reduce disability and improve the quality of life of those affected by brain diseases"
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The Gap
� Neurodegener ati ve diseases
(NDDs) are estimated
to contribute as much as 35%
of the disease burden
in the seven major
pharmaceuti cal markets
� By the year 2040, NDDs will
become the world’s second
leading cause of death, overtaking
cancer (WHO)
� Despite these statistics,
there are few effective treatments
for the majority of the Central
Nervous System (CNS) disorders
� Lack of translation
of promising therapies
from experimental models
into clinical applications
The blood-brain barrier is a major impediment
in the CNS drug development
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Medicinal
chemistryStrategies to overcome
the blood-br ain barrierDirect injection
BBB
CNS Drug Development
More that 98% of small drugs and virtually all biologics
(peptides, antibodies, genes) do not cross the BBB
in pharmacologically relevant concentrations
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The Blood-Brain Barrier
and CNS Drug Development
Dr. Danica Stanimirovic
The screen versions of these slides have full details of copyright and acknowledgements 2
BBB Disruption
Sledge and hammer approachBrick wall
Blood-Brain Barrier
Ligands
Pumps
Channels
Pores
‘Intelligent’ drug deliveryFluid, specialized membrane
Brain endothelial cells
The Blood-Brain Barrier –
Evolving Concept
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Neurovascular Unit –
an Integrated Concept
The BBB properties result from interactions and functional
integration of brain endothelial cells and other cellular
and acellular components of the neurovascular unit
Endothelialcells
Pericyte
Tight Junction (TJ)
Astrocyte footprocesses
Neuron
Basementmembrane
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Tight Junctions – the ‘Physical’ Barrier
Signaling roles of TJ proteins
Break Modulate
Cadherins Cadherins
ZO1
ZO2
Actin
Claudins
Occludin
Junctional adhesion molecule
Tight junction
Adherens junction
Apical plasma membrane
Basal plasma membrane
Paracellular Space
Shin K et al., Annu Rev Cell Dev Biol 22:207-35 20066
The Blood-Brain Barrier
and CNS Drug Development
Dr. Danica Stanimirovic
The screen versions of these slides have full details of copyright and acknowledgements 3
Zhang W, Stanimirovic D; (2005) The Transport Systems of the Blood-Brain Barrier, In: The Blood-Brain Barrier and its Microenvironment: Basic Physiology to Neurological Disease (deVries E and Prat A, eds), Taylor & Francis Group, NY
Transporters: Dynamic Blood-Brain Barrier
Endothelial cells
Pericyte
Tight Junction (TJ)
Transporters
for glucose, amino acids,nucleosides
Oatp2MCT1MRP-1?MRP-5?
Transportersfor glucose, amino acids,nucleosides,MCT1, Oatp2,Oatp14, OCTN2
Physical barrier (TJ):passive diffusion
for water, fluorescein,lipid soluble small
molecules, Amphiphilic drugs
AME
RME
Efflux pumps:MDR-1 Pgp
MRP-1, ABCG2,OAT3
Neurotransmittertransporters
Circulation
Brain
EAAT1-3Oatp2ATA2
GAT2/BGT-1OAT
Astrocyte foot processes
Neuron
Basement membrane
Transferrin receptor
Insulin receptorLRP,SR
RAGE(-)(+)
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Strategies to Deliver Drugs to the Brain
Trans-cranial
� Intra-cerebroventricular (ICV) or intrathecal injection
� Intra-cerebral (IC) injection/implantation
� Convection-enhanced diffusion (CED)
Non-Vascular Routes
Some advantages
� Region selective delivery
� Cells, biologics
� No systemic toxicity
Disadvantages
� Invasive
� Side effects
� Limited transfer/diffusion
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The subarachnoid space (D) is continuous with the extracellular space of the olfactory mucosa; From Mathison et al., (1998)
� Olfactory (trans-nasal) route
Strategies to Deliver Drugs to the Brain
Non-Vascular Routes
� The capacit y of the system in humans?
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The Blood-Brain Barrier
and CNS Drug Development
Dr. Danica Stanimirovic
The screen versions of these slides have full details of copyright and acknowledgements 4
� 100 billion capillaries in human brain
� Surface area – 20 m2
� Diffusion to target – 10-25 m
Trans-vascular (BBB) routes
Strategies to Deliver Drugs to the Brain
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Routes for Drug Delivery Across the BBB
De Boer AG, Gaillard PJ. 2007, Annu rev pharmacol, Toxicol 47:323-55 11
1. Transcellular Lipophilic Diffusion
For many drugs/solutes
(open points), there is a clear
correlation between lipid
solubility and BBB penetration
Octanol/wat er partition coefficient
Facilitated transport
Efflux transporters
Adapted from Levin (1980)
Lipidization strategies
� Increase of non-specific
distribution in all organs
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The Blood-Brain Barrier
and CNS Drug Development
Dr. Danica Stanimirovic
The screen versions of these slides have full details of copyright and acknowledgements 5
2. Inhibition of Efflux Pumps
50%-70% of drugs are substrates of MDR-1 Pgp and BCRP
[11C]Carasolol
+Cyclosporin Control
Elsinga P et al., Mol Imaging Biol (2005) 7:37Y44
Examples of increased
CNS drug deliv ery
with Pgp/BCRP inhibitors:
PSC833: Vinblastine
Paclitaxel
GF120918: Colchicine
Vinblastin
Amprenivir
LY335979: Paclitaxel
Nelfinavir
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3. Paracellular Hydrophilic Diffusion
BBB disruption
BBB disruption has serious long-term consequences
Osmotic BBB disruption
� Intracarotid injection of hy pertonic solution of manitol
�Hospitalization
Focal ultrasound or electromagnetic f ield
�Focused, local BBB disruption
Transient chemical modulation of TJ tightness
�Bradiky nin B(2) receptor agonist, RMP-7 (Cereport) [400D 1kD]
�Alky lgly cerols
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4. Carrier-Mediated Transport
�The GLUT1 glucose transporter transports glucose and other hexoses,
including 2-deoxyglucose and fluoro-deoxyglucose used in (PET) scanning
�LAT1 transports large and small neutral amino acids, as well as certain amino
acid drugs including l-dopa, α-methyl-dopa, α-methyl-para-tyrosine or gabapentin
�The cationic amino acid transporter CAT1 transports basic amino acids,
such as arginine or lysine
�The monocarboxylic acid transporter MCT1 transports lactate, pyruvate,
ketone bodies and certain monocarboxylic acid drugs such as probenecid
�The concentrative nucleoside transporter CNT2 transports purine nucleosides,
and certain pyrimidine nucleosides such as uridine
�Choline undergoes carrier-mediated transport across the BBB
via a sodium-independent process
�The purine bases, but not the pyrimidine bases, undergo carrier-mediated
transport across the BBB
The various CMT systems provide a diverse space of molecular
structure that could be mimicked with medicinal chemistry
modifications of drugs that normally do not cross the BBB
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The Blood-Brain Barrier
and CNS Drug Development
Dr. Danica Stanimirovic
The screen versions of these slides have full details of copyright and acknowledgements 6
RCA-1
5. Adsorptive Endocytosis
� Cationized IgG
� Cationized liposomes
� Cell-penetrating peptide v ectors (Sy nB1 and penetratin)
Glycocalyx
++
+Drug
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6. Receptor-Mediated Transcytosis
ReceptorLigand
� Clathrin-coated v esicles
� Cav eolae
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Molecular ‘Trojan Horses’ Concept
Pardridge WM: Curr Opin Pharmacol 2006 Oct;6(5):494-500 18
The Blood-Brain Barrier
and CNS Drug Development
Dr. Danica Stanimirovic
The screen versions of these slides have full details of copyright and acknowledgements 7
‘Trojan Horses’
‘Receptors-targets’
Transferrin receptor
Insulin receptor (IGFR1 & 2)
LRP1/2
Targeting vectors
OX-26 (IgG – rat; mouse)
IR-Ab (mouse, humanized)
ApoE (nanocarriers)
Angio-pepTM
Mellanotransf err in (p97)
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Pardridge WM: Pharmaceutical Research (2007)
Human Insulin Receptor (HIR)
as a ‘Trojan Horse’ for Drug Delivery to Brain
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Poly(butyl cyanoacrylate) nanoparticles coated with polysorbate 80 (Tween 80), which further binds Apo-E in the bloodstream
‘Trojan Horses’ and Nanocarriers
LRP
Kreuter J (2001) Adv Drug Deliv Rev 47(1):65-81 21
The Blood-Brain Barrier
and CNS Drug Development
Dr. Danica Stanimirovic
The screen versions of these slides have full details of copyright and acknowledgements 8
‘Trojan Horses’ - Considerations
�Selectiv ity of receptor
f or brain v essels
�Capacity /saturation
�Regulation/recy cling
�Distribution in brain v ascular beds
�Modulation in disease
Receptor Targeting vectors
� Selectiv ity to receptor
� Binding af f inity
� Versatility – link
and release strategies
� Brain ef f lux (FcRn)
� Pharmacokinetics, stability,
non-specif ic clearance
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Discovery and Development of Novel
‘Trojan Horses’
Discovery Platforms
� Genomics/proteomics –
selective ‘biomarkers’ of brain vessels
� Phage-display platforms
single-domain antibodies
single chain antibodies
peptides
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IgG
150 kDa
VHH
13 kDa
scFv
25 kDa
Fab
50 kDa
Single Domain Antibodies
� Recognition of uncommon and hidden epitopes
� Cav ity binding – enzy mes and receptors
� Innov ativ e drug f ormats
� Ease of manuf acture and f ormulation
� Solubility and stability
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The Blood-Brain Barrier
and CNS Drug Development
Dr. Danica Stanimirovic
The screen versions of these slides have full details of copyright and acknowledgements 9
Single-Domain Antibody
Phage Display Libraries
Platform technology for selecting ‘targeting’ antibodies
against diseased tissues (e.g., cancer, vasculature, etc.)
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Selection of the BBB – Targeting sdAbs
Biopanning & Enrichment
Subtractive panning
between human lung
and human brain
endothelial cells
Selecting species
from enriched library
that transmigrate across
the BBB in vitro
Test BBB permeability
in vivo
Tanha et al., Methods in Mol Medicine, 2003Muruganandam et al., FASEB J 16:240 26
Brain-Targeting Single-Domain Antibodies
Two sdAb that cross the BBB model in vitro, FC5 and FC44,
were selected from non-immunized llama sdAb library
Muruganandam et al., FASEB J 16:240 27
The Blood-Brain Barrier
and CNS Drug Development
Dr. Danica Stanimirovic
The screen versions of these slides have full details of copyright and acknowledgements 10
Mechanisms of Transport
� Mediated by clathrin-coated v esicles
� Saturable and energy dependent
� Triggered by FC5 binding
to the sialogly coprotein polarized
to the luminal surf ace of HCEC
FC5/Clathrin
Receptor-Mediated Transcytosis (RMT)
Abulrob et al., J Neurochemistry, 2005
FC5 co-localizes with clathrin-coated vesicles
Human brain endothelial cells
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HRP
HRP
Linking Molecules to FC5
Develop chemistries to link moieties to sdAbs
0 10 20 30 40 50 60 705
10
15
20
25
30
35
Transport across BBB in vitro
Time [min]
Clearance [ml/] HRP-IgG-cysFC5
HRP-IgG
cysFC5
IgG cysFC5
IgG
+
Abulrob et al., Drug Transporters and the Diseased Brain, ICS 1277 (2005) 29
In Vivo Biodistribution of FC5
eXplore Optix shown with optional user workstation
� ‘Real-time’
observ ation of drug
biodistribution in vivo
Time-domain, near infra-red in vivo optical imaging
+
Cy5.5
FC5-
Conjugation of FC5 with NIR probe Cy5.5
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The Blood-Brain Barrier
and CNS Drug Development
Dr. Danica Stanimirovic
The screen versions of these slides have full details of copyright and acknowledgements 11
Improving Pharmacokinetics
PEGylation increased plasma
half-life of FC5 from ~5 min to ~4 h !
� PEG-ylat ed di-FC5
cysactivated
cys
PEG
Maleimide
FC5 Cy5.5lys
FC5 lys Cy5.5
Site-specific PEGylation
� Pentameric FC5 (P5)
Oligomerization
Pentamerization increases
efficiency of transcytosis
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NC11
FC5
P5
diFC5-PEG
Cy5.5
Brain Accumulation of FC5 Formulations
Head Imaging, 6h
Grids - 5 mm
Volume concentration slices
Volume concentration planes
Dose: 3 nM of sdAb i.v. 32
Detection of FC5 in Brain Tissues
� At the end of imaging protocol – animals injected
with Tomato lectin (green) to stain brain vessels
� Brain and other organs dissected and sectioned
� Sections stained for cell-selective antigens
(GFAP-astrocytes; NeuN-neurons, CD31-endothelial
cells; laminin – basement membrane)
� Cy5.5-FC5 co-localized with above markers
by confocal microscopy
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The Blood-Brain Barrier
and CNS Drug Development
Dr. Danica Stanimirovic
The screen versions of these slides have full details of copyright and acknowledgements 12
Detection of FC5 in the Brain
by Confocal Microscopy
FCFCFCFC5555 NCNCNCNC11111111
Green – vessels
Red – Cy5.5 (FC5 or NC11)
Ve sselVe sselVe sselVe ssel
Laminin (BM)
CD31
FC5
Dose: 3 nM of sdAb i.v.Immunostaining – 6 h after injection 34
FC5 (green) NeuN (red) Cell nuclei (blue)
FC5
Frontal cortex
FC5, double injection (0, 2 h)
immunostaining – 4 h after last injection
Brain vessel
Neurons
Detection of FC5 in the Brain
by Confocal Microscopy
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Summary
� FC5 transmigrates the BBB after intravenous injection
� FC5 is detected by confocal microscopy in both brain
vessels and brain parenchyma
� PEGylation and oligomerization of FC5 improves
its brain biodistribution
� FC5 is a novel ‘Trojan horse’ for BBB drug delivery
Can FC5 carry therapeutic into the brain?
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The Blood-Brain Barrier
and CNS Drug Development
Dr. Danica Stanimirovic
The screen versions of these slides have full details of copyright and acknowledgements 13
FC5-Functionalized Liposomes
Mol ratio
Lecithin (758) 2
Cholesterol (387) 1
DOGS-NTA (1057.02) 0.06
dode-PE (889.29) 0.01
mPEG-DSPE (2805.54) 0.08
Composition of liposomes
for doxorubicin encapsulation
Lecithin
Cholesterol
DOGS-NTA
DSPE-PEG2000
FC5 or P5
Dox
Size 137 nm, SD = 59 nm (41%)
Doxorubicin is substrate for P-glycoprotein
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Delivery of Doxorubicin to the Brain
Using FC5-Functionalized Liposomes
Optical imaging 24h after injection
Lipo- FC5
Lipo- P5
Lipo- NC11
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Separate capillaries
and brain parenchy ma
Measure Dox
concentrations using
f luorometric methods
Integrated imaging/delivery platform
Delivery of Doxorubicin to the Brain
Using FC5-Functionalized Liposomes
Af ter imaging - perf usion
Dissect brains
*
*
# *
# *
*
Capillaries
Brain
0
50
100
150
200
250
300
350
400
Dox-lipo-
FC5
Dox-lipo-P5 Dox-lipo Free Dox
ng doxorubicin/ g tissue
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The Blood-Brain Barrier
and CNS Drug Development
Dr. Danica Stanimirovic
The screen versions of these slides have full details of copyright and acknowledgements 14
‘Tailoring’ BBB Delivery Strategies
to Specific Brain Diseases
� The immature or aged BBB
� The local vs. generalized brain delivery
� Acute (trauma, stroke) vs. chronic brain diseases
(AD, neurodegeneration)
� The level and timing of the BBB disruption
in various diseases
� Molecular and cellular targets within brain
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� Side effects and acceptability of protocols
� Pharmacologically-relevant doses
� Clinical and commercial viability
� Tailoring both delivery system and delivered therapeutic
‘Tailoring’ BBB Delivery Strategies
to Specific Brain Diseases
The brain vasculature and the BBB
are still vastly unexploited ‘targets’
for the development of CNS therapies
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